Use of an agent that restores tissue perfusion and oxygenation

ABSTRACT

The presently disclosed subject matter provides methods for increasing perfusion in hypoxic regions of tissues in subjects. Also provided are methods for treating diseases and/or disorders associated with hypoxia in subjects, methods for increasing sensitivity of tumors to radiation and/or chemotherapy treatments, methods for delaying tumor growth in subjects, and methods for inhibiting tumor blood vessel growth in subjects. In some embodiments, the presently disclosed methods involve administering to subjects in need thereof a first composition selected from the group consisting of a nitrosylated hemoglobin and an agent that induces nitrosylation of endogenous hemoglobin in the subject and a second composition comprising a hyperoxic gas. In some embodiments, the presently disclosed methods also include treating a tumor with radiation therapy, chemotherapy, photodynamic therapy, immunotherapy, or combinations thereof. Also provided are inhalable gases that can be employed in the presently disclosed methods.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/404,494, filed Apr. 13, 2006, now U.S. Pat. No. 7,338,670 whichitself claims the benefit of U.S. Provisional Patent Application Ser.No. 60/671,179, filed Apr. 14, 2005; the disclosure of each of which isincorporated herein by reference in its entirety.

GOVERNMENT INTEREST

The presently disclosed subject matter was made with United StatesGovernment support under Grant No. CA40355 awarded by the NationalInstitutes of Health/National Cancer Institute. Thus, the U.S.Government has certain rights in the presently disclosed subject matter.

TECHNICAL FIELD

The presently disclosed subject matter provides methods for increasingperfusion in hypoxic regions of tissues in subjects. Also provided aremethods for treating diseases and/or disorders associated with hypoxiain subjects, methods for increasing sensitivity of tumors to radiationand/or chemotherapy treatments, and/or other circulating antitumortreatments, diagnostic agents, and/or prognostic agents, methods fordelaying tumor growth in subjects, and methods for inhibiting tumorblood vessel growth in subjects. Also provided are compositions that canbe employed in the presently disclosed methods.

BACKGROUND

Hypoxia (a reduction in the normal level of oxygen tension) is a commonfeature of both experimental and human solid tumors. It results from animbalance between oxygen supply and consumption (Dachs & Tozer, 2000).This fundamental characteristic of tumor cells is of major clinicalimportance since hypoxia can predict both tumor progression and poortreatment outcome (Dachs & Tozer, 2000; Vaupel et al., 2001).

Diffusion-limited hypoxia is generally believed to arise from theincreasing metabolic demands of the growing mass of cells at increasingintercapillary distances. However, there is now clear evidence that thismight not be the only determinant of chronic hypoxia. Another cause isof hypoxia relates to the level of oxygenation of the incoming blood.Indeed, before entering a tumor, a continuous diffusion of oxygenbetween the blood and the interstitium along the vascular tree accountsfor an estimated two-thirds hemoglobin (Hb) deoxygenation (Pittman,1995). Then, in order for blood to reach the tumor periphery, it mustfirst pass through moderately hypoxic tissues where most of theremaining oxygen in the blood is extracted (Dewhirst et al., 1999).

As a result of this steep vascular gradient of hemoglobin desaturation,many vessels in tumors carry severely deoxygenated blood, so theirability to supply oxygen to the tumor is limited. Additional regions ofsevere hypoxia in solid tumors result from the uneven partition oferythrocytes in the tumor microvasculature, leading to measurablechanges in vascular and perivascular pO₂ (fluctuant hypoxia; Dewhirst etal., 1996; Kimura et al., 1996; Hill et al., 1996). Low-frequency redcell flux and pO₂ fluctuations in tumors are of high magnitude, whichresults in a decrease in average tissue oxygenation and a greatervariability in local tissue pO₂ (Braun et al., 1999; Kimura et al.,1996; Tsai & Intaglietta, 1993). Fluctuating arteriolar diameter mightalso contribute to flow instability in tumor microvessels (Dewhirst etal., 1996). This, along with the tortuous tumor vasculature accountingfor unstable vascular pressures, results in very unstable blood flow,unstable and heterogeneous oxygenation, and areas of fluctuant hypoxiain tumors. Local tumor pO₂ can often transiently drop below 3-10 mm Hg(Kimura et al., 1996; Braun et al., 1999), which is considered to be thecritical pO₂ for radiosensitization (radiobiological hypoxia).

Thus, radiological treatment of tumors is often met with limited successdue at least in part to a sub-optimal concentration of oxygen in thetumors. In biological systems, irradiation induces water radiolysis andthe subsequent production of the highly reactive reactive oxygen species(OH., O₂.⁻, and H₂O₂; Mundt et al., 2000). Their most importantreactions with biological structures, in terms of therapeutic effect,are those involving DNA, because they are more likely to impair cellsurvival. They lead to the reversible formation of DNA radicals, whichcan lead to strand breaks. However, if oxygen is present, then it canreact with DNA to produce DNA-O₂., which then undergoes further reactionto ultimately yield DNA-OOH (Horsman & Overgaard, 2002).Oxygen-dependent fixation of DNA damage is known as the ‘oxygen effect’.It accounts for the high radiosensitivity of oxygenated areas in tumors;by contrast, hypoxic areas are less sensitive. Upon base damage, DNAreorganization can result in intra- or inter-strand crosslinking,crosslinks between DNA and chromosomal proteins, and single or doubleDNA strand breaks (McMillan & Steel, 2002). As a consequence,radiation-induced damage is primarily manifested by the loss of cellularreproductive integrity.

Many chemotherapeutic agents are also dependent on cellular oxygenationfor maximal efficacy. Cytotoxic alkylating agents, such as the nitrogenmustard alkylating agent melphalan, comprise a class of chemotherapeuticdrugs that act by transferring alkyl groups to DNA during cell division.Following this, the DNA strand breaks or cross-linking of the twostrands occurs, preventing subsequent DNA synthesis. In a study byTeicher et al., tumor cells in normoxic conditions were more sensitiveto melphalan, in contrast to their hypoxic counterparts (Teicher et al.,1985). Under hypoxic conditions, alkylating agents might have lessefficacy due to the increased production of nucleophilic substances suchas glutathione that can compete with the target DNA for alkylation(Hamilton et al., 2002).

Other examples of drugs directly effected by a lack of O₂ include theantibiotic bleomycin and the podophyllotoxin derivative etoposide.Bleomycin does not have maximum efficacy due to the reduced generationof free radicals under hypoxic conditions (Teicher et al., 1981).Etoposide efficacy is reduced due to free radical scavengers,dehydrogenase inhibitors, and dehydrogenase substrates, which preventthe formation of single-strand breaks, thereby decreasing the cytotoxiceffects of etoposide (Kagan et al., 2001).

Anticancer drugs such as alkylating agents and antimetabolites actmainly during DNA synthesis by causing damage to the DNA and initiatingapoptosis. These drugs can therefore have reduced efficacy on slowlycycling tumor cells under hypoxic conditions. DNA-damagingchemotherapeutic agents such as alkylating agents and platinum compoundsmight also have compromised function due to increased activity of DNArepair enzymes under hypoxic conditions. Hypoxia also increases theproduction of various proteins that appear to be responsible for drugresistance (Goldstein, 1996; Zhong et al., 1999; Kinoshita et al., 2001;Comerford et al., 2002).

What are needed, then, are new methods and compositions that can beemployed for reducing and/or eliminating hypoxic regions of tumors andother cells that are generally treated with therapies that require thepresence of oxygen to be maximally effective. The presently disclosedsubject matter addresses this and other needs in the art.

SUMMARY

The presently disclosed subject matter provides methods of increasingperfusion in a hypoxic region of a tissue in a subject. In someembodiments, the methods comprise administering to the subject (a) acomposition selected from the group consisting of a nitrosylatedhemoglobin, an agent that induces nitrosylation of hemoglobin in thesubject, a hemoglobin and an agent that induces nitrosylation ofhemoglobin in the subject, and combinations thereof; and (b) a hyperoxicgas. In some embodiments, the agent that induces nitrosylation ofhemoglobin in the subject comprises ethyl nitrite (ENO). In someembodiments, the ethyl nitrite (ENO) is administered to the subject asan inhalable composition comprising about 100 parts per million (ppm) inthe hyperoxic gas. In some embodiments, the hemoglobin is present withina red blood cell. In some embodiments, the red blood cell is presentwithin the subject. In some embodiments, the hemoglobin is presentwithin a red blood cell that is administered to the subject. In someembodiments, the hyperoxic gas is selected from the group consisting ofpure oxygen and carbogen. In some embodiments, the tissue comprises atumor cell, a cancer cell, and combinations thereof. In someembodiments, the subject is a mammal. In some embodiments, the mammal isa human. In some embodiments, the administering results increases a pO₂value in at least a fraction of the hypoxic region of the tissue to atleast about 10 mm Hg. In some embodiments, the hypoxic region of thetissue results from a disease or disorder in the subject, and theadministering ameliorates at least one symptom associated with thedisease or disorder in the subject.

The presently disclosed subject matter also provides methods fortreating a disease or disorder associated with hypoxia in a subject. Insome embodiments, the methods comprise administering to the subject (a)a composition selected from the group consisting of a nitrosylatedhemoglobin, an agent that induces nitrosylation of hemoglobin in thesubject, a hemoglobin and an agent that induces nitrosylation ofhemoglobin in the subject, and combinations thereof; and (b) a hyperoxicgas. In some embodiments, the agent that induces nitrosylation ofendogenous hemoglobin in the subject comprises ethyl nitrite (ENO). Insome embodiments, the ethyl nitrite (ENO) is administered to the subjectat about 100 parts per million (ppm) in the hyperoxic gas. In someembodiments, the hemoglobin is present within a red blood cell. In someembodiments, the red blood cell is present within the subject. In someembodiments, the hemoglobin is present within a red blood cell that isadministered to the subject. In some embodiments, the hyperoxic gas isselected from the group consisting of pure oxygen and carbogen. In someembodiments, the disease or disorder comprises a tumor, a cancer,peripheral vascular disease, diabetes, a disease related to smoking,cirrhosis, rheumatoid arthritis, stroke, myocardial infarction, andcombinations thereof. In some embodiments, the disease or disordercomprises a tumor, a cancer, or combinations thereof, and the methodsfurther comprise treating the subject with a second therapy selectedfrom the group consisting of radiotherapy, chemotherapy, immunotherapy,surgery, photodynamic therapy, and combinations thereof. In someembodiments, the treating the subject with a second therapy step isperformed concurrently with the administering step. In some embodiments,the subject is a mammal. In some embodiments, the mammal is a human. Insome embodiments, the administering results increases a pO₂ value in atleast a fraction of the hypoxic region of the tissue to at least about10 mm Hg.

The presently disclosed subject matter also provides methods forincreasing a sensitivity of a tumor in a subject to a treatment. In someembodiments, the methods comprise administering to the subject (a) acomposition selected from the group consisting of a nitrosylatedhemoglobin, an agent that induces nitrosylation of hemoglobin in thesubject, a hemoglobin and an agent that induces nitrosylation ofhemoglobin in the subject, and combinations thereof; and (b) a hyperoxicgas, wherein the administering increases pO₂ in a plurality of cells ofthe tumor to above about 10 mm Hg, thereby increasing sensitivity of thetumor to the treatment. In some embodiments, the treatment is selectedfrom the group consisting of radiotherapy, chemotherapy, photodynamictherapy, immunotherapy, and combinations thereof. In some embodiments,the agent that induces nitrosylation of endogenous hemoglobin in thesubject comprises ethyl nitrite (ENO). In some embodiments, the ethylnitrite (ENO) is administered to the subject as an inhalable compositioncomprising about 100 parts per million (ppm) in the hyperoxic gas. Insome embodiments, the administering comprises administering a minimallytherapeutic dose of the first composition and the second composition. Insome embodiments, the tumor is resistant to radiation therapy,chemotherapy, or both radiation therapy and chemotherapy. In someembodiments, the hemoglobin is present within a red blood cell. In someembodiments, the red blood cell is present within the subject. In someembodiments, the hemoglobin is present within a red blood cell that isadministered to the subject. In some embodiments, the subject is amammal. In some embodiments, the mammal is a human.

The presently disclosed subject matter also provides methods fordelaying tumor growth in a subject. In some embodiments, the methodscomprise (a) administering to the subject (i) a composition selectedfrom the group consisting of a nitrosylated hemoglobin, an agent thatinduces nitrosylation of hemoglobin in the subject, a hemoglobin and anagent that induces nitrosylation of hemoglobin in the subject, andcombinations thereof; and (ii) a hyperoxic gas, wherein theadministering increases pO₂ in a plurality of cells of the tumor toabove about 10 mm Hg; and (b) treating the tumor with radiation therapy,chemotherapy, or both radiation therapy and chemotherapy, whereby tumorgrowth in the subject is delayed. In some embodiments, the tumor isresistant to radiation therapy, chemotherapy, or both radiation therapyand chemotherapy. In some embodiments, the agent that inducesnitrosylation of endogenous hemoglobin in the subject comprises ethylnitrite (ENO). In some embodiments, the ethyl nitrite (ENO) isadministered to the subject as an inhalable composition comprising about100 parts per million (ppm) in the hyperoxic gas. In some embodiments,the hemoglobin is present within a red blood cell. In some embodiments,the red blood cell is present within the subject. In some embodiments,the hemoglobin is present within a red blood cell that is administeredto the subject. In some embodiments, the subject is a mammal. In someembodiments, the mammal is a human. In some embodiments, the treatingthe tumor with radiation therapy comprises treating the tumor with asubtherapeutic dose of ionizing radiation. In some embodiments, thetreating the tumor with chemotherapy comprises administering to thesubject a therapeutically effective amount of a chemotherapy agent. Insome embodiments, the methods further comprise promoting tumorregression.

The presently disclosed subject matter also provides methods forinhibiting tumor blood vessel growth in a subject. In some embodiments,the methods comprise (a) administering to the subject (i) a compositionselected from the group consisting of a nitrosylated hemoglobin, anagent that induces nitrosylation of hemoglobin in the subject, ahemoglobin and an agent that induces nitrosylation of hemoglobin in thesubject, and combinations thereof; and (ii) a hyperoxic gas, wherein theadministering increases pO2 in a plurality of cells of the tumor toabove about 10 mm Hg; and (b) treating the tumor with radiation therapy,chemotherapy, or both radiation therapy and chemotherapy, whereby tumorblood vessel growth in the subject is inhibited. In some embodiments,the agent that induces nitrosylation of hemoglobin in the subjectcomprises ethyl nitrite (ENO). In some embodiments, the ethyl nitrite(ENO) is administered to the subject as an inhalable compositioncomprising about 100 parts per million (ppm) in the hyperoxic gas. Insome embodiments, the hemoglobin is present within a red blood cell. Insome embodiments, the red blood cell is present within the subject. Insome embodiments, the hemoglobin is present within a red blood cell thatis administered to the subject. In some embodiments, the subject is amammal. In some embodiments, the mammal is a human. In some embodiments,the methods further comprise delaying tumor growth in the subject. Insome embodiments, the methods further comprise promoting tumorregression in the subject.

The presently disclosed subject matter also provides methods ofenhancing delivery of a diagnostic, therapeutic, or prognostic agent toa tumor in a subject. In some embodiments, the methods comprise (a)administering to the subject a composition selected from the groupconsisting of a nitrosylated hemoglobin, a nitrosylating agent thatinduces nitrosylation of hemoglobin in the subject, a hemoglobin and anitrosylating agent that induces nitrosylation of hemoglobin in thesubject, and combinations thereof; and (b) administering a diagnostic,therapeutic, or prognostic agent to the subject, wherein delivery of thediagnostic, therapeutic, or prognostic agent agent to a tumor in thesubject is enhanced. In some embodiments, the composition comprises thediagnostic, therapeutic, or prognostic agent. In some embodiments, thediagnostic, therapeutic, or prognostic agent comprises an imaging agent.In some embodiments, the methods further comprise administering to thesubject a hyperoxic gas selected from the group consisting of 100%oxygen and carbogen. In some embodiments, the subject is a mammal. Insome embodiments, the mammal is a human.

The presently disclosed subject matter also provides inhalablecompositions. In some embodiments, the inhalable compositions comprise(a) a composition selected from the group consisting of a nitrosylatedhemoglobin, an agent that induces nitrosylation of hemoglobin in thesubject, a hemoglobin and an agent that induces nitrosylation ofhemoglobin in the subject, and combinations thereof; and (b) a hyperoxicgas. In some embodiments, the inhalable composition comprises at leastabout 100 parts per million (ppm) ethyl nitrite (ENO). In someembodiments, the hyperoxic gas is selected from the group consisting ofpure oxygen and carbogen.

Accordingly, it is an object of the presently disclosed subject matterto provide methods and compositions for increasing perfusion in ahypoxic region of a tissue in a subject. This and other objects areachieved in whole or in part by the presently disclosed subject matter.

An object of the presently disclosed subject matter having been statedabove, other objects and advantages of the presently disclosed subjectmatter will become apparent to those of ordinary skill in the art aftera study of the following description and non-limiting Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict time course plots of oxygenated hemoglobin (oxy-Hb)-and S-nitrosylated hemoglobin (SNO-Hb)-induced decreases in tumor bloodflow. Changes in perfusion of muscle and tumors were determined byinserting Laser Doppler probes into the quadriceps muscle or tumors ofrats breathing room air. FIG. 1A depicts perfusion of the quadricepsmuscle. At time 0, animals were infused i.v. with albumin (∘, N=3),oxy-Hb (●, N=4), or SNO-Hb (▪, N=7). FIG. 1B depicts tumor perfusion inrats infused i.v. with oxy-Hb (●, N=5, n=10) or albumin (∘, N=3, n=3).FIG. 1C depicts tumor perfusion in rats infused i.v. with SNO-Hb (▪,N=7, n=13), or SNO-albumin (□, N=7, n=14). “N” refers to the number ofanimals per group and “n” refers to the number of individualmeasurements. *p<0.05; **p<0.01 between t=0 and t=20 minutes (two-wayANOVA).

FIG. 2 is a bar graph depicting stabilization of the SNO moiety onplasma SNO-Hb by hyperoxic breathing in vivo. The concentration of SNOproteins was determined over time in the plasma of rats following i.v.infusion of SNO-Hb. The half-life of SNO-Hb is shown for rats breathingroom air or 100% O₂. (N=5). **p<0.01 (Student's t test).

FIGS. 3A and 3B depict selective modulation of the effects of SNO-Hb ontumor perfusion during oxygen breathing. Changes in tumor perfusion weredetermined by inserting Laser Doppler probes into rat tumors. FIG. 3A isa graph depicting relative tumor perfusion in rats infused i.v. withSNO-Hb with the rats breathing room air (▪, N=7, n=13) or 100% O₂ (□,N=7, n=13). FIG. 3B is a graph depicting relative tumor perfusion inrats infused i.v. with Oxy-Hb with the rats breathing room air (●, N=5,n=10) or 100% O₂ (◯, N=4, n=8). Where indicated, O₂ breathing wasadministered from t=−5 minutes (arrowheads) to the end of theexperiment; infusions were started at t=0. ***p<0.005 between t=0 andt=20 minutes (two-way ANOVA).

FIGS. 4A-4F are plots depicting an unmasking of systemic pressoractivity of plasma SNO-Hb in rats breathing room air or 100% oxygen.SNO-Hb or oxy-Hb was infused i.v. into rats at t=0. Where indicated,rats breathed 100% O₂ from t=−5 minutes (arrowheads) until the end ofthe experiment. FIG. 4A depicts a time course of the vasoactive effectsof SNO-Hb on tumor feeding arterioles determined from window chambervideo analyses in rats breathing room air (▪, N=4) or 100% O₂ (□, N=6).FIG. 4B depicts changes in muscle perfusion detected by Laser Dopplerprobes placed in the quadriceps muscle of tumor-bearing rats afterSNO-Hb infusion. Rats breathed room air (▪, N=7) or 100% O₂ (□, N=7). InFIG. 4B, *p<0.05 between t=0 and t=20 minutes (two-way ANOVA). FIG. 4Cdepicts changes in the mean arterial pressure (MAP) and FIG. 4D depictschanges in the heart rate (HR) following SNO-Hb infusion. Rats breathedroom air (▪, N=7) or 100% O₂ (□, N=7). FIG. 4E depicts changes in MAPand FIG. 4F depicts in HR following oxy-Hb infusion. Rats breathed roomair (●, N=5) or 100% O₂ (◯, N=5). In FIGS. 4C-4F, *p<0.05 at theindicated time points versus values at t=0 (Student's t test).

FIGS. 5A-5D are plots depicting the preservation of the pressor activityof SNO-Hb by intra-arterial infusion, which also accounts for loss ofO₂-dependence. SNO-Hb was infused i.v. or i.a. in rats at t=0. Whereindicated, rats breathed O₂ from t=−5 minutes (arrowheads). FIG. 5Adepicts changes in perfusion simultaneously determined using LaserDoppler probes in rat tumors, and FIG. 5B depicts changes in perfusionsimultaneously determined using Laser Doppler probes in the quadricepsmuscle. In FIG. 5A, SNO-Hb was infused i.v. in animals breathing roomair (▪, N=7, n=13) or 100% O₂ (□, N=7, n=14), or i.a. with room air (▴,N=7, n=14) or 100% O₂ (Δ, N=7, n=13). Note the loss of O₂-dependence fori.a. infusions. In FIG. 5B, SNO-Hb was infused i.a. in animals breathingroom air (▴, N=7) or 100% O₂ (Δ, N=6). FIG. 5C depicts changes in themean arterial pressure (MAP) and FIG. 5D depicts changes in the heartrate (HR) following i.a. infusion of SNO-Hb. Rats breathed room air (▴,N=7) or 100% O₂ (Δ, N=7). In FIGS. 5C and 5D, *p<0.05 versus values att=0 for the indicated time points (Student's t test).

FIGS. 6A-6D are plots depicting measurements of muscle or tumorperfusion in rats breathing 100% O₂ or 100% O₂ plus 100 parts permillion ethyl nitrite (ENO). The first arrow indicates the start of thegas/gas mixture delivery, and the second arrow indicates the time atwhich the gas/gas mixture delivery is stopped. FIG. 6A is a graphdepicting changes in pO₂ (mm Hg) in quadriceps muscle before, during,and after breathing 100% O₂ (□) or 100% O₂ plus 100 parts per millionENO (▪). FIG. 6B is a graph depicting changes in pO₂ (mm Hg) inquadriceps muscle before, during, and after breathing room air plus 100parts per million ENO (□). FIG. 6C is a graph depicting changes in pO₂(mm Hg) in a tumor before, during, and after breathing 100% O₂ (□) or100% O₂ plus 100 parts per million ENO (▪). FIG. 6D is a graph depictingchanges in pO₂ (mm Hg) in a tumor before, during, and after breathingroom air plus 100 parts per million ENO (□). Note that in rats breathing100 ppm ENO plus 100% O₂ the pO₂ increased from less than 10 mm Hg togreater than 10 mm Hg, but the same increase was not seen in tumors ofrats breathing ENO under normoxic conditions.

FIG. 7 is a graph depicting tumor growth delay in rats breathing 100 ppmENO plus 100% O₂ after treatment with radiation. Tumors implanted inrats were irradiated daily with 2 Gy of irradiation on days 0-4.Relative tumor volumes were determined on the indicated days in ratsthat breathed room air (Δ, N=4), 100 O₂ (▴, N=4), or 100 ppm ENO plus100% O₂ (▪). Also included are relative tumor volumes for rats thatbreathed room air (◯, N=3) and were not treated with radiation.

DETAILED DESCRIPTION

The most powerful sensitizer for radiotherapy is oxygen, and an agentwith similar radiosensitizing properties is nitric oxide. As disclosedherein, a vasodilating agent is used to increase perfusion and maintainoxygenation of tumors during high oxygen content gas breathing. Thevasodilating agent is capable of carrying NO to the tumor site andreleases NO in a form that can increase radiosensitivity and/orchemosensitivity of the tumor at least in part by reducing tumor hypoxiaand by providing NO in sufficient quantities to mimic oxygen in fixationof sublethal radiation or chemotherapy damage.

The steal effect arises as a consequence of the non tumor-selectivevasoresponses to vasodilators. It prevents the clinical use of theseagents for sensitizing tumors to radiation therapy and chemotherapy(Feron, 2004). Ethyl nitrite (ENO; also referred to as O-nitrosoethanol)has not been previously tested in this regard. ENO is a gas that can bedelivered through airways to subjects and it does not reduce bloodpressure. As such, it can react with fully-oxygenated hemoglobin (Hb)within red blood cells that transit through the lung vasculature,leading to the formation of S-nitrosylated Hb (SNO-Hb).

SNO-Hb is used to maintain perfusion and improve oxygenation of tumorsduring high oxygen content gas breathing. During hyperoxic gasbreathing, nitrosohemoglobin releases NO in the distal arterioles,thereby opposing the vasoconstricting effects of hyperoxia. This effectimproves delivery of oxygen and perfusion to tumor regions that mightotherwise be hypoxic. Additionally, this vasodilating agent is capableof releasing NO in tumor regions in a form that can increaseradiosensitivity and/or chemosensitivity of hypoxic regions by providingNO in sufficient quantities to mimic oxygen in fixation of sublethalradiation or chemotherapy damage, following exposure. Thus, thepresently disclosed subject matter includes the combination of improvedoxygen delivery and NO delivery to increase tumor radiosensitivityand/or chemosensitivity.

SNO-Hb is delivered to the tumor via red blood cells. The deliveryoccurs by having a tumor-bearing subject breathe a NO donor gas (such asethyl nitrite) that is mixed with a hyperoxic gas, such as pure oxygenor carbogen (95% oxygen, 5% CO₂). The addition of hyperoxic gasbreathing maintains Hb in the R-state, which prevents unloading of O₂and SNO until the Hb enters into tumor regions, which are relativelyhypoxic as compared to normal tissues. This approach maintains orimproves perfusion and oxygen delivery to relatively hypoxic tumorregions.

I. Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a tumor cell” includes aplurality of such tumor cells, and so forth.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration, or percentage ismeant to encompass variations of in some embodiments, ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, andin some embodiments ±0.1% from the specified amount, as such variationsare appropriate to perform the disclosed methods.

As used herein, “significance” or “significant” relates to a statisticalanalysis of the probability that there is a non-random associationbetween two or more entities. To determine whether or not a relationshipis “significant” or has “significance”, statistical manipulations of thedata can be performed to calculate a probability, expressed as a “pvalue”. Those p values that fall below a user-defined cutoff point areregarded as significant. In some embodiments, a p value less than orequal to 0.05, in some embodiments less than 0.01, in some embodimentsless than 0.005, and in some embodiments less than 0.001, are regardedas significant. Accordingly, a p value greater than or equal to 0.05 isconsidered not significant.

As used herein, the term “subject” refers to any organism for whichapplication of the presently disclosed subject matter would bedesirable. The subject treated in the presently disclosed subject matterin its many embodiments is desirably a human subject, although it is tobe understood that the principles of the presently disclosed subjectmatter indicate that the presently disclosed subject matter is effectivewith respect to all vertebrate species, including mammals, which areintended to be included in the term “subject”. Moreover, a mammal isunderstood to include any mammalian species in which treatment of atumor and/or a cancer is desirable, particularly agricultural anddomestic mammalian species.

More particularly provided is the treatment of mammals such as humans,as well as those mammals of importance due to being endangered (such asSiberian tigers), of economic importance (animals raised on farms forconsumption by humans) and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans (such ascats and dogs), swine (pigs, hogs, and wild boars), ruminants (such ascattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also provided is the treatment of birds, including the treatmentof those kinds of birds that are endangered, kept in zoos, as well asfowl, and more particularly domesticated fowl, i.e., poultry, such asturkeys, chickens, ducks, geese, guinea fowl, and the like, as they arealso of economic importance to humans. Thus, contemplated is thetreatment of livestock, including, but not limited to, domesticatedswine (pigs and hogs), ruminants, horses, poultry, and the like.

As used herein, the term “cell” is used in its usual biological sense.In some embodiments, the cell is present in an organism, for example,mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, cats,and rodents. In some embodiments, the cell is a eukaryotic cell (e.g., amammalian cell, such as a human cell). The cell can be of somatic orgerm line origin, totipotent or pluripotent, dividing or non-dividing.The cell can also be derived from or can comprise a gamete or embryo, astem cell, or a fully differentiated cell.

The term “tumor” as used herein encompasses both primary andmetastasized solid tumors and carcinomas of any tissue in a subject,including, but not limited to breast; colon; rectum; lung; oropharynx;hypopharynx; esophagus; stomach; pancreas; liver; gallbladder; bileducts; small intestine; urinary tract including kidney, bladder andurothelium; female genital tract including cervix, uterus, ovaries(e.g., choriocarcinoma and gestational trophoblastic disease); malegenital tract including prostate, seminal vesicles, testes and germ celltumors; endocrine glands including thyroid, adrenal, and pituitary; skin(e.g., hemangiomas and melanomas), bone or soft tissues; blood vessels(e.g., Kaposi's sarcoma); brain, nerves, eyes, and meninges (e.g.,astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,neuroblastomas, Schwannomas and meningiomas). The term “tumor” alsoencompasses tumors arising from hematopoietic malignancies such asleukemias, including chloromas, plasmacytomas, plaques and tumors ofmycosis fungoides and cutaneous T-cell lymphoma/leukemia, and lymphomasincluding both Hodgkin's and non-Hodgkin's lymphomas. The term “tumor”also encompasses radioresistant and/or chemoresistant tumors, including,but not limited to radioresistant and/or chemoresistant variants of theany of the tumors listed above.

The terms “radiosensitivity” and “radiosensitive”, as used herein todescribe a tumor, refer to a quality of susceptibility to treatmentusing ionizing radiation. Thus, radiotherapy can be used to delay growthof a radiosensitive tumor. Radiosensitivity can be quantified bydetermining a minimal amount of ionizing radiation that can be used todelay tumor growth. Thus, the term “radiosensitivity” can refer to aquantitative range of radiation susceptibility.

The terms “sensitivity to chemotherapy”, “chemosensitivity”, and“chemosensitive”, as used herein to describe a tumor, refer to a qualityof susceptibility to treatment using chemotherapy. Thus, chemotherapycan be used to delay growth of a tumor sensitive to chemotherapy.Sensitivity of chemotherapy can be quantified by determining a minimaldosage of chemotherapy that can be used to delay tumor growth. Thus, thephrase “sensitivity to chemotherapy” can refer to a quantitative rangeof chemotherapy susceptibility.

The terms “radiation resistant tumor” and “radioresistant tumor” eachgenerally refer to a tumor that is measurably less responsive toradiotherapy than are other tumors. Representative radiation resistanttumor models include glioblastoma multiforme and melanoma. Similarly,the terms “chemotherapy resistant tumor” and “chemoresistant tumor”generally refer to a tumor and/or to a tumor region that is measurablyless responsive to chemotherapy than are other tumors and/or tumorregions.

The term “delaying tumor growth” refers to an increase in duration oftime required for a tumor to grow a specified amount. For example,treatment with the compositions and/or methods disclosed herein candelay the time required for a tumor to increase in volume by a specifiedfraction (e.g., 2-fold, 3-fold, etc.) from an initial day of measurement(day 0), and/or can refer to an increase in the time required for thetumor to grow to a certain volume (e.g., 1 cm³). Alternatively or inaddition, the term “delayed” (and grammatical variants thereof in thecontext of tumor growth can refer to a decrease in the rate at which atumor grows and/or the rate at which individual cells of a tumorproliferate. As such, tumor growth delay in some embodiments can beconsidered relative to how the same tumor and/or tumor cell would havegrown in the absence of treatment with the methods and/or compositionsdisclosed herein.

The term “increase,” as used herein to refer to a change inradiosensitivity and/or sensitivity to chemotherapy of a tumor, refersto change that renders a tumor more susceptible to treatment by ionizingradiation and/or chemotherapy. Alternatively stated, an increase inradiosensitivity and/or chemosensitivity can refer to a decrease in theminimal amount of ionizing radiation and/or chemotherapy thateffectively delays tumor growth. An increase in radiosensitivity and/orchemosensitivity can also comprise delayed tumor growth when acomposition of the presently disclosed subject matter is administeredwith radiation and/or chemotherapy as compared to a same dose ofradiation and/or chemotherapy alone. In some embodiments, an increase inradiosensitivity and/or chemosensitivity refers to an increase of atleast about 2-fold, in some embodiments an increase of at least about5-fold, and in some embodiments an increase of at least 10-fold. In someembodiments of the presently disclosed subject matter, an increase inradiosensitivity and/or chemosensitivity comprises a transformation of aradioresistant and/or chemoresistant tumor to a radiosensitive and/orchemosensitive tumor.

The term “tumor regression” generally refers to any one of a number ofindices that suggest change within the tumor to a less developed form.Such indices include, but are not limited to a destruction of tumorvasculature (for example, a decrease in vascular length density or adecrease in blood flow), a decrease in tumor cell survival, a decreasein tumor volume, and/or a decrease in tumor growth rate. Methods forassessing tumor growth delay and tumor regression are known to one ofordinary skill in the art.

II. Compositions

The presently disclosed subject matter provides in some embodimentscompositions comprising one or more of a nitrosylated hemoglobin, anagent that induces nitrosylation of hemoglobin in the subject, ahemoglobin and an agent that induces nitrosylation of hemoglobin in thesubject, and combinations thereof. As such, the compositions disclosedherein are designed in some embodiments to deliver to, or produce in, asubject a nitrosylated hemoglobin.

Thus, in some embodiments the compositions disclosed herein comprise ahemoglobin that has been nitrosylated prior to administration to asubject. Any nitrosylation methods can be used to nitrosylate thehemoglobin to be administered, and include, but are not limited to themethods disclosed in the Materials and Methods used in EXAMPLES 1-5presented hereinbelow. Thus, a hemoglobin or hemoglobin substitute canbe modified in vitro or in vivo prior to administration to a subject. Insome embodiments, the hemoglobin is present within a red blood cell.

Additionally, in some embodiments the compositions disclosed hereincomprise an agent that induces nitrosylation of hemoglobin in thesubject. As used herein, the phrase “an agent that induces nitrosylationof hemoglobin in a subject” refers to any agent that when administeredto a subject results in a higher level of hemoglobin nitrosylation inthe subject than would have been present in the subject in the absenceof the agent. A representative agent that induces nitrosylation ofhemoglobin in a subject comprises a nitric oxide donor.

In some embodiments, a nitric oxide donor comprises ethyl nitrite (ENO).It is understood, however, that other NO donors can be employed in thepractice of the presently disclosed subject matter, with the provisothat the NO donor is capable of nitrosylating hemoglobin in vitro and/orin vivo, optionally endogenous hemoglobin present within a red bloodcell. Other NO donors include, but are not limited toS-nitrosoglutathione (GSNO) and ethyl nitrate (ENO₂).

Additionally, the route of administration of the agent (e.g., a NOdonor) is not to be viewed as a limitation of the presently disclosedsubject matter. Therefore, while in some embodiments an NO donor isprovided in a breathable gas, NO donors can also be administered forexample in an oral form, in the form of intravenous, intra-arterial,intramuscular, subcutaneous, or other injectable form, provided that theadministration of the NO donor results in an increased level ofnitrosylation of hemoglobin in the subject (e.g., the subject's ownhemoglobin and/or an administered hemoglobin or hemoglobin product).

In some embodiments, the agent that induces nitrosylation of hemoglobinin the subject is capable of nitrosylating hemoglobin present within ared blood cell that is already in the subject. The red blood cell can beone of the subject's own red blood cells or can be a red blood cell thatwas administered to the subject.

In some embodiments, the compositions further comprise a hyperoxic gas.As used herein, the phrase “hyperoxic gas” refers to a gas thatcomprises an oxygen content that is greater than that found in normalroom air (i.e., about 21%). Thus, a “hyperoxic gas” is a gas thatincludes, for example, about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, or 25% oxygen. In some embodiments, ahyperoxic gas is 100% oxygen, and in some embodiments a hyperoxic gas iscarbogen (about 95% oxygen, about 5% carbon dioxide).

Breathing hyperoxic gas can lead to vasoconstriction, which cannegatively impact the ability of the hyperoxic gas to deliver increasedoxygen to a target tissue (e.g., a region of hypoxia). Accordingly, thehyperoxic gas is mixed in some embodiments with a vasodilation agentsuch as nitric oxide and/or a nitric oxide donor including, but notlimited to an alkyl nitrite such as C1-C6 straight chain, branched, orsubstituted alkyl nitrite (e.g., ethyl nitrite). While applicants do notwish to be bound by any particular theory of operation, a nitric oxidedonor is chosen that is capable of nitrosylating hemoglobin either invitro or in vivo, including hemoglobin that is within a red blood cellor not within a red blood cell.

III Methods of Treatment

III.A. Methods for Increasing Perfusion

The presently disclosed subject matter provides in some embodimentsmethods for treating conditions associated with hypoxia. As used herein,the phrase “condition associated with hypoxia” refers to any diseaseand/or disorder, and/or any symptom thereof, which results from and/oris exacerbated or aggravated by hypoxia. Exemplary conditions associatedwith hypoxia include, but are not limited to tumors, cancers, peripheralvascular diseases, diabetes, diseases related to smoking, cirrhosis,rheumatoid arthritis, stroke, myocardial infarction, and combinationsthereof. Accordingly, the methods and compositions disclosed herein canbe employed to treat and/or ameliorate at least one symptom of theseconditions.

As disclosed herein, certain diseases and/or disorders associated withhypoxia, and/or one or more symptoms thereof, can be treated by reducinghypoxic regions of relevant tissues in a subject. This can beaccomplished in any manner including, but not limited to delivering moreoxygen to the tissue such as by increasing perfusion in a hypoxic regionof the tissue.

Accordingly, the presently disclosed subject matter provides in someembodiments methods for increasing perfusion in a hypoxic region of atissue in a subject. In some embodiments, the methods compriseadministering to the subject an inhalable composition comprising ethylnitrite (ENO) and optionally a hyperoxic gas.

Increasing perfusion can also lead to other beneficial outcomes. Forexample, it is also expected that the distribution of other agentsdelivered by the blood to a tumor would be enhanced using the methodsand compositions disclosed herein. Exemplary, non-limiting agentsinclude diagnostic, therapeutic, and/or prognostic agents such ascontrast agents using for functional imaging of tumors.

III.B. Methods for Treating Tumors and/or Cancers

The presently disclosed subject matter also provides methods fortreating subjects with particular conditions associated with hypoxia. Insome embodiments, a condition associated with hypoxia is a tumor and/ora cancer. In some embodiments, the methods and compositions disclosedherein are part of a combination therapy as discussed in more detailhereinbelow.

In some embodiments, the presently disclosed subject matter relates tomethods for delaying tumor growth in a subject. In some embodiments, themethods comprise administering to the subject a composition selectedfrom the group consisting of a nitrosylated hemoglobin, an agent thatinduces nitrosylation of hemoglobin in the subject, a hemoglobin and anagent that induces nitrosylation of hemoglobin in the subject, andcombinations thereof, optionally in combination with a hyperoxic gas. Insome embodiments, the administering increases pO₂ in a plurality ofcells of the tumor to above about 10 mm Hg, whereby tumor growth in thesubject is delayed. In some embodiments, the hyperoxic gas is selectedfrom the group consisting of pure oxygen and carbogen. In someembodiments, the administering step increases pO₂ in a plurality ofcells of the tumor to above about 10 mm Hg. In some embodiments, pO₂ ismonitored in the tumor in real time to ensure that it is at least about10 mm Hg. In some embodiments, the methods also comprise treating thetumor with radiation therapy, chemotherapy, or both radiation therapyand chemotherapy.

Additionally, the presently disclosed subject matter relates in someembodiments to methods for inhibiting tumor blood vessel growth in asubject. In some embodiments, the methods comprise administering to thesubject an inhalable composition comprising ethyl nitrite (ENO) andoptionally a hyperoxic gas (for example, a hyperoxic gas selected fromthe group consisting of pure oxygen and carbogen). In some embodiments,the administering step increases pO₂ in a plurality of cells of thetumor to above about 10 mm Hg. In some embodiments, the methods furthercomprise and (b) treating the tumor with radiation therapy,chemotherapy, or both radiation therapy and chemotherapy. As usedherein, such an inhibition need not be absolute, but can include adecrease in a rate and/or extent of tumor angiogenesis that results fromemploying the methods and/or compositions disclosed herein.

IV. Methods for Enhancing Delivery of Diagnostic. Therapeutic, and/orPrognostic Agents

The presently disclosed subject matter also provides methods forenhancing delivery of a diagnostic, therapeutic, or prognostic agent toa target tissue including, but not limited to a tumor, in a subject. Insome embodiments, the methods comprise (a) administering to the subjecta composition selected from the group consisting of a nitrosylatedhemoglobin, a nitrosylation agent that induces nitrosylation ofhemoglobin in the subject, a hemoglobin and a nitrosylation agent thatinduces nitrosylation of hemoglobin in the subject, and combinationsthereof; and (b) administering the diagnostic, therapeutic, orprognostic agent to the subject, wherein delivery of the diagnostic,therapeutic, or prognostic agent to a tumor in the subject is enhanced.The compositions and methods disclosed herein can be employed toincrease perfusion and/or blood flow in target tissues including, butnot limited to hypoxic regions of tumors. In some embodiments, themethods further comprise administering to the subject a hyperoxic gasselected from the group consisting of 100% oxygen and carbogen.

Accordingly, the compositions and methods disclosed herein can beemployed to enhance delivery of diagnostic, therapeutic, and prognosticagents that are carried via the bloodstream (e.g., agents that areinjected intravenously) by enhancing blood flow in the target tissue.The delivery of any such agent can be enhanced, including but notlimited to therapeutic agents such as drugs and diagnostic and/orprognostic agents such as imaging agents. Particularly with respect toimaging agents that cannot be conveniently administered directly to atarget tissue, the ability to increase the delivery of the imaging agentby employing the compositions and methods disclosed herein can result ingreater capacity to image the target tissue, less time the subject mustspend in the imaging apparatus, a lower dose of imaging agent that isrequired for acceptable imaging, and combinations thereof. Particularlywith respect to imaging agents that have known toxicity associated withtheir use, the ability to use less of the agent can be a considerableadvantage.

V. Combination Therapies

Tumors and/or cancers can be treated using combination therapiescomprising combinations of surgery, radiotherapy, and/or chemotherapy,and/or other therapies include, but not limited to photodynamic therapy(PDT) and immunotherapy (IT). Thus, the presently disclosed subjectmatter can be employed as a part of a combination therapy. As usedherein, the phrase “combination therapy” refers to any treatment whereinthe methods and compositions disclosed herein are used in combinationwith another therapy including, but not limited to radiation therapy(radiotherapy), chemotherapy, surgical therapy (e.g., resection), PDT,IT, and combinations thereof.

As disclosed herein, various therapies that are employed to treatneoplastic disease can be relatively ineffective if the tumor and/orcancer includes localized regions of hypoxia. This is based at least inpart on the requirement for the therapy to generate free radicals fromoxygen, which does not occur in hypoxic sites.

As a result, the methods and/or compositions disclosed herein can beemployed to enhance the effectiveness of a second treatment such asradiotherapy, chemotherapy, photodynamic therapy, immunotherapy, andcombinations thereof. In these embodiments, the methods relate toincreasing the sensitivity of a tumor and/or a tumor cell in a subjectto a treatment, and in some embodiments the methods compriseadministering to the subject an inhalable composition comprising ethylnitrite (ENO) and optionally a hyperoxic gas, which further optionallycan be selected from the group consisting of pure oxygen and carbogen,whereby pO₂ in a plurality of cells of the tumor is increased to aboveabout 10 mm Hg, thereby increasing sensitivity of the tumor to thesecond treatment (e.g., a treatment selected from the group consistingof radiotherapy, chemotherapy, photodynamic therapy, immunotherapy, andcombinations thereof).

V.A Radiation Treatment

In some embodiments, the methods and compositions disclosed herein areemployed in a combination therapy with radiation treatment. For suchtreatment of a tumor, the tumor is irradiated concurrent with, orsubsequent to, administration of an inhalable composition as disclosedherein. One of skill in the medical art can design, upon considerationof the instant disclosure, an appropriate dosing schedule for treating asubject with radiation in conjunction with the compositions and methodsdisclosed herein. For example, tumors can be irradiated withbrachytherapy utilizing high dose rate or low dose rate brachytherapyinternal emitters.

In order to enhance the benefit gained from administration of thecompositions disclosed herein, the timing of administration of thecomposition and the radiation treatment should be adjusted such that thepO₂ in the tumor to be treated is at least about 10 mm Hg during atleast a portion of the entire period when the radiation is beingadministered (optionally, during the entire period). Accordingly, thecomposition can be administered beginning, for example, 5, 10, 15, 20,30, 45, or 60 minutes before the radiation treatment is administered.Additionally, the subject can continue to breathe the composition whilethe radiation treatment is being administered. Upon cessation of theradiation treatment, the administration of the composition can also beterminated.

It is understood that since radiotherapy typically is repeated severaltimes in order to affect a maximal response, the administration of thecomposition can likewise be repeated each time radiotherapy is given.Thus, the time course over which a inhalable composition as disclosedherein is administered can comprise in some embodiments a period ofseveral weeks to several months coincident with radiotherapy, but insome embodiments can extend to a period of 1 year to 3 years as neededto effect tumor control. Alternatively, a composition can beadministered prior to an initial radiation treatment and then at desiredintervals during the course of radiation treatment (e.g., weekly,monthly, or as required).

Subtherapeutic or therapeutic doses of radiation can be used fortreatment of a tumor and/or a cancer as disclosed herein. In someembodiments, a subtherapeutic or minimally therapeutic dose (whenadministered alone) of ionizing radiation is used. For example, the doseof radiation can comprise in some embodiments at least about 2 Gyionizing radiation, in some embodiments about 2 Gy to about 6 Gyionizing radiation, and in some embodiments about 2 Gy to about 3 Gyionizing radiation. When radiosurgery is used, representative doses ofradiation include about 10 Gy to about 20 Gy administered as a singledose during radiosurgery or about 7 Gy administered daily for 3 days(about 21 Gy total). When high dose rate brachytherapy is used, arepresentative radiation dose comprises about 7 Gy daily for 3 days(about 21 Gy total). For low dose rate brachytherapy, radiation dosestypically comprise about 12 Gy administered twice over the course of 1month. ¹²⁵I seeds can be implanted into a tumor can be used to deliververy high doses of about 110 Gy to about 140 Gy in a singleadministration.

Radiation can be localized to a tumor using conformal irradiation,brachytherapy, stereotactic irradiation, intensity modulated radiationtherapy (IMRT), and/or can be localized to a tumor by employing vectorsthat comprise, but are not limited to, proteins, antibodies, liposomes,lipids, nanoparticles, and combinations thereof. The threshold dose fortreatment can thereby be exceeded in the target tissue but avoided insurrounding normal tissues. For treatment of a subject having two ormore tumors, local irradiation enables differential drug administrationand/or radiotherapy at each of the two or more tumors. Alternatively,whole body irradiation can be used, as permitted by the low doses ofradiation required following radiosensitization of the tumor.

Radiation can also comprise administration of internal emitters, forexample ¹³¹I for treatment of thyroid cancer, NETASTRON™ and QUADRAGEN®pharmaceutical compositions (Cytogen Corp., Princeton, N.J., UnitedStates of America) for treatment of bone metastases, ³²P for treatmentof ovarian cancer. Other internal emitters include ¹²⁵I, iridium, andcesium. Internal emitters can be encapsulated for administration or canbe loaded into a brachytherapy device.

Radiotherapy methods suitable for use in the practice of presentlydisclosed subject matter can be found in Leibel & Phillips, 1998, amongother sources.

V.B. Chemotherapy Treatment

In some embodiments, the methods and compositions disclosed herein areemployed in a combination therapy with chemotherapy. Particularchemotherapeutic agents are generally chosen based upon the type oftumor to be treated, and such selection is within the skill of themedical professional.

Chemotherapeutic agents are generally grouped into several categoriesincluding, but not limited to DNA-interactive agents, anti-metabolites,tubulin-interactive agents, hormonal agents, and others such asasparaginase or hydroxyurea. Each of the groups of chemotherapeuticagents can be further divided by type of activity or compound. For adetailed discussion of various chemotherapeutic agents and their methodsfor administration, see Dorr et al., 1994, herein incorporated byreference in its entirety.

In order to reduce the mass of the tumor and/or stop the growth of thecancer cells, a chemotherapeutic agent should prevent the cells fromreplicating and/or should interfere with the cell's ability to maintainitself. Exemplary agents that accomplish this are primarily theDNA-interactive agents such as Cisplatin, and tubulin interactiveagents.

DNA-interactive agents include, for example, alkylating agents (e.g.,Cisplatin, Cyclophosphamide, Altretamine); DNA strand-breakage agents(e.g., Bleomycin); intercalating topoisomerase II inhibitors (e.g.,Dactinomycin and Doxorubicin); non-intercalating topoisomerase IIinhibitors (e.g., Etoposide and Teniposide); and the DNA minor groovebinder Plicamycin.

Generally, alkylating agents form covalent chemical adducts withcellular DNA, RNA, and/or protein molecules, and with smaller aminoacids, glutathione, and/or similar biomolecules. These alkylating agentstypically react with a nucleophilic atom in a cellular constituent, suchas an amino, carboxyl, phosphate, or sulfhydryl group in nucleic acids,proteins, amino acids, or glutathione.

Anti-metabolites interfere with the production of nucleic acids byeither of two major mechanisms. Some of the drugs inhibit production ofdeoxyribonucleoside triphosphates that are the immediate precursors forDNA synthesis, thus inhibiting DNA replication. Some of the compoundsare sufficiently like purines or pyrimidines to be able to substitutefor them in the anabolic nucleotide pathways. These analogs can then besubstituted into the DNA and RNA instead of their normal counterparts.

Hydroxyurea appears to act primarily through inhibition of the enzymeribonucleotide reductase.

Asparaginase is an enzyme which converts asparagine to nonfunctionalaspartic acid and thus blocks protein synthesis in the tumor.

Tubulin interactive agents act by binding to specific sites on tubulin,a protein that polymerizes to form cellular microtubules. Microtubulesare critical cell structure units. When the interactive agents bind onthe protein, the cell can not form microtubules. Tubulin interactiveagents include Vincristine and Vinblastine, both alkaloids andPaclitaxel.

Adrenal corticosteroids are derived from natural adrenal cortisol orhydrocortisone. They are used because of their anti-inflammatorybenefits as well as the ability of some to inhibit mitotic divisions andto halt DNA synthesis. These compounds include Prednisone,Dexamethasone, Methylprednisolone, and Prednisolone.

The hormonal agents and leutinizing hormones are not usually used tosubstantially reduce the tumor mass. However, they can be used inconjunction with the chemotherapeutic agents. Hormonal blocking agentsare also useful in the treatment of cancers and tumors. They are used inhormonally susceptible tumors and are usually derived from naturalsources. These include, but are not limited to estrogens and conjugatedestrogens, progestins, and androgens. Leutinizing hormone releasinghormone agents or gonadotropin-releasing hormone antagonists are usedprimarily the treatment of prostate cancer. These include leuprolideacetate and goserelin acetate. They prevent the biosynthesis of steroidsin the testes. Other anti-hormonal agents include anti-estrogenicagents, anti-androgen agents, and anti-adrenal agents such as Mitotaneand Aminoglutethimide.

VI. Other Oxygen-dependent Treatments

Various other oxygen-dependent therapies can be employed that would beexpected to benefit from the presently disclosed compositions andmethods. Examples of therapies that could benefit from a combinedtreatment regimen employing the methods and/or compositions disclosedherein include any antitumor therapies wherein an active agent reachesthe tumor from the systemic circulation. Such therapies include, but arenot limited to, photodynamic therapy, hormone therapy, immunotherapy,gene therapy, antivascular therapy, antiangiogenic therapy, cell therapy(based on injection or mobilization of cells with an antitumoractivity), and combinations thereof, including any of these therapiesfurther in combination with radiotherapy and/or chemotherapy.Circulating antitumor agents for which therapeutic efficacy can beincreased through administration of the compounds disclosed hereininclude ions, small molecules, macromolecules, peptides, proteins,nucleotides, virus, liposomes, emulsions, bacteria, immune cells, stemcells, and combination (s) thereof.

One further therapy that can benefit from the methods and compositionsdisclosed herein is photodynamic therapy (PDT). The therapeutic effectof PDT is highly dependent upon oxygen availability. PDT involves twoindividual inactive components that are combined to induce cellular andtissue effects in an O₂-dependent manner (Dolmans et al., 2003). Thefirst component is the photosensitizer (e.g., porphyrins), whichlocalizes to the tumor. The second component involves the administrationof light of a specific wavelength that activates the photosensitizer. Insitu, the activated photosensitizer transfers energy from light to O₂ togenerate reactive oxygen species, which then mediate cellular toxicity.The biological responses are activated only in the particular areas oftissue that have been exposed to light and contain sufficient amounts ofO₂. The toxic species formed with PDT is singlet oxygen. Thus, thecytotoxic effects of PDT drugs are entirely O₂-dependent andphotosensitization typically does not occur in hypoxic tumor areas. As aresult, increasing the local concentration of oxygen in a tissuetargeted for PDT (e.g., a hypoxic region of a tumor) can be expected toenhance the efficacy of the PDT in that target tissue.

Another approach to tumor and/or cancer treatment is immunotherapy.Immunotherapy generally relates to strategies designed to augment theability of the subject's immune system to recognize tumor cells andeliminate them. Typically, these strategies are intended to boost and/orto activate antitumor lymphocytes. A non-limiting example ofimmunotherapy relates to the use of therapeutic vaccines. Nakedpeptides, peptides loaded on protein carriers, and/or antigen presentingcells loaded with peptide can also elicit an antitumor response in vivoin subjects. Responses generally involve the activation of antitumor Tlymphocytes, but also can include activation of other immunomodulatorycells including, but not limited to memory lymphocytes, natural killercells, and B lymphocytes.

Heterologous or autologous immune transfer can also be a part of anantitumor treatment strategy, and all these approaches are expected tobenefit from the presently disclosed subject matter because immune cellsenter the tumor through the systemic circulation and need oxygen tolive, to multiply, and to act in a hypoxic environment. See e.g., U.S.Pat. No. 5,405,940 and progeny thereof (including, but not limited toU.S. Pat. Nos. 5,462,871; 5,695,994; 6,034,214; 6,222,012; 6,379,901;and 6,488,932); PCT International Patent Application Publications WO94/05304; WO 94/16713; WO 95/25530; WO 95/33855; WO 96/29409; WO98/32855; and WO 98/58956; van Baren et al. 2005; Godelaine et al.,2003; Chaux et al., 1999; Van den Eynde et al., 1995; van der Bruggen etal., 1994; Gaugler et al., 1994; Boon et al., 1994; Traversari et al.,1992; and other patents, published patent applications, and scientificpublications from Dr. Thierry Boon and co-workers (each of which isincorporated by reference herein in its entirety) for discussion of MAGEand MAGE-related approaches to antitumor therapy, several of which arecurrently in clinical trials.

Other immunotherapeutic strategies are designed to affect the anti-tumoractivities of the subject's macrophages, which are frequently found inclose association with tumors (e.g., so-called “tumor-associatedmacrophages (TAMs); see Lewis & Murdoch, 2005 for a review). In somecases, macrophages can comprise up to 80% of the cell mass in certaintumors (see Bingle et al., 2002).

Macrophage recruitment to tumors results in alterations in the tumormicroenvironment, and is a strongly negative predictive factor foroutcome. For example, it has been shown that hypoxic areas of tumorsattract macrophages and macrophage precursors (Murdoch et al., 2004),and that the macrophage response to hypoxia can actually increase theability of the tumor cells to proliferate and/or metastasize (see Lewis& Murdoch, 2005, and references therein). Macrophage responses tohypoxia include the production of various growth factors relevant totumor cell proliferation and angiogenesis (e.g., epidermal growthfactor, vascular endothelial growth factor) as well as the production ofimmunomodulatory factors such as prostaglandin E₂ and IL-10 that candownregulate the anti-tumor response of various immune effector cells.

Additionally, hypoxia inhibits the phagocytosis of tumor cells and othernecrotic cells by macrophages. Taken together, therefore, it is clearthat hypoxia alters macrophage biological activities in ways that aloneor in combination can severely negatively impact the ability of thesubject's immune system to respond to the presence of tumor and/orcancer cells.

EXAMPLES

The following Examples have been included to illustrate modes of thepresently disclosed subject matter. In light of the present disclosureand the general level of skill in the art, those of skill willappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter.

Materials and Methods Used in Examples 1-6

Rats and Tumors. Female Fischer 344 rats (Charles River Laboratories,Raleigh, N.C., United States of America), bearing or not bearing thesyngeneic mammary adenocarcinoma R3230AC (Hilf et al., 1965), were usedin all experiments. Tumor pieces from donor rats were implanted inanesthetized animals (100 mg/Kg ketamine and 10 mg/Kg xylazine i.p.).Tumors were grown in the subcutis of the left lateral quadriceps muscleor, for window chamber experiments, between two fascial layers on theback of rats. Animals were then randomly assigned to treatment groups.Further interventions/observations were performed on anesthetizedanimals (50 mg/Kg pentobarbital i.p.) maintained at 37° C. on atemperature-controlled thermal blanket. Where indicated, some rats werefitted with a facemask for breathing 100% O₂. All experiments wereapproved by the Duke University Institutional Animal Care and UseCommittee.

Drugs. SNO-Hb was synthesized through reaction of purified humanhemoglobin A₀ (Apex Bioscience, Durham, N.C., United States of America)with SNO-cysteine in non-acidic conditions as described in McMahon &Stamler, 1999. Solutions of purified human hemoglobin A₀ (ApexBioscience, Durham, N.C., United States of America) were dialyzedovernight at 4° C. against 2% (w/v) aerated sodium tetraborate pH 9.2,0.5 mmol/L EDTA. For SNO-Hb synthesis, L-cysteine hydrochloride (0.55mol/L in 0.5 mol/L HCl, 0.5 mol/L EDTA) was first reacted with an equalvolume of 0.5 mol/L NaNO₂ to yield Cys-NO. Then, a 10-fold molar excessof Cys-NO was reacted with dialyzed oxy-Hb at a 1:20 (v/v) ratio for 10minutes. The reaction was terminated by centrifugation through a fineSephadex G-25 chromatography column (Pharmacia Biotech, Uppsala, Sweden)equilibrated with PBS, pH 7.4, 0.5 mmol/L EDTA. Under these conditions,the yield of oxy-Hb S-nitrosylation was consistently 1.9S-nitroso-cysβ93 per tetramer.

SNO-albumin was synthesized by reacting an equal volume of bovine serumalbumin (0.2 mmol/L in HCl 0.1 mmol/L, 0.5 mmol/L EDTA) with 0.2 mmol/LNaNO₂ for 30 minutes. All solutions (diluted at 200 μmol/L in PBS pH7.4, 0.5 mmol/L EDTA) were kept on ice or frozen and protected fromlight until administration. Less than 2-weeks old solutions were infusedat a dose of 200 nmol/Kg in 0.25 ml, immediately followed by a 0.25 mldelivery of saline. Assuming a plasma volume of about 3 ml per 100 g inrats and no significant partition in blood cells, this dose wouldachieve a maximum plasma concentration of about 6.5 μmol/L for alldrugs. To avoid volume-induced alterations of hemodynamics, solutionswere infused at 0.5 ml/minutes from time 0.

Surgery. For i.v. infusions, the femoral artery and vein were cannulatedand drugs were infused through the venous cannula. For i.a. infusions,the femoral and left carotid arteries were cannulated and drugs wereinfused through the carotid cannula. For window chamber experiments, thetwo window frames were surgically positioned in the dorsal skin flap,followed by tumor cell transplantation to the chamber tissue, asdescribed in Dewhirst et al., 1996. Following several days of tumorgrowth, direct visualization of tumor-feeding arterioles was performedusing intravital microscopy.

Blood Gas and Hemoglobin Saturation Measurements in Tumor Microvessels.Blood gases and Hb oxygen saturation were measured using a 1640 oximetercoupled to a 482 co-oximeter (Instrumentation Laboratories, Lexington,Mass., United States of America) from 0.5 ml samples of femoral arterialand venous blood. For tumor microvessels in window chambers, Hbsaturation was calculated from transmission optical measurements ofvascular absorbance using a hyperspectral imaging technique described inSorg et al., 2005. A liquid crystal tunable filter (CRI, Inc., Woburn,Mass., United States of America) placed in front of a CCD camera (DVCCompany, Austin, Tex., United States of America) was used forband-limited imaging.

Blood Flow. Blood flow was measured using Laser Doppler flowmetry. Forflank tumor experiments, 300 μm diameter Laser Doppler probes (OxiFlo,Oxford Optronix, Oxford, United Kingdom) were simultaneously insertedinto the tumor and quadriceps muscle of each animal. For window chamberexperiments, Laser Doppler probes (LASERFLO®, TSI, St. Paul, Minn.,United States of America) were positioned beneath the tumor window asdescribed in Dunn et al., 1999.

SNO-Hb Assay. The determination of SNO-Hb concentrations was based on areaction where cleavage of S-nitrosothiols yields a nitrosant thatactivates 4,5-diaminofluorescein (DAF-2, Sigma, St. Louis, Mo., UnitedStates of America). Briefly, blood samples were collected in heparinizedtubes just before infusion and at 1 minute, 5 minutes, and every 5minutes after infusion of SNO-Hb i.v. for 30 minutes. Plasma (100 μl)was immediately mixed with DAF-2 (30 μM final concentration). Half ofthis solution was then reacted for 10 minutes with an equal volume of1.2 mM HgCl₂. The reaction was terminated by centrifugation in filtertubes (Nanosep, Pall, East Hills, N.Y., United States of America). Uponlight excitation at 485 nm, DAF-2-associated light emission was read at520 nm. SNOs were quantified as the difference in fluorescence signalsgenerated in the presence and absence of HgCl₂, which specificallycleaves SNOs, yielding nitrite. Data acquired before drug infusionallowed basal SNO levels to be determined. The plasma half-life ofSNO-Hb was calculated from exponential decay curves fitting eachexperimental curve. Hb concentration in plasma samples was determined byvisible spectrophotometry.

Vascular Reactivity In Vivo. Tumor-feeding arterioles in window chamberswere visualized using transillumination. Arteriolar diameters weremeasured from videotaped images using an image-shearing monitor (IPMInc., San Diego, Calif., United States of America).

Mean Arterial Pressure and Heart Rate. Mean arterial pressure (MAP) andheart rate (HR) were measured with a blood pressure analyzer (Digi-Med,Micro-Med, Louisville, Ky., United States of America) connected to thefemoral artery cannula as described in Dewhirst et al., 1996.

Statistical Analyses. All values are shown in the Figures as means ±S.E.Time curves were normalized to the baseline before infusion. “N” refersto the number of animals per group, and “n” to individual measurements.Repeated-measure two-way ANOVA or Student's t tests were used asindicated.

Example 1 SNO-Hb and Oxy-Hb i.v. Reduce Muscle and Tumor Perfusion inNormoxic Rats

Whether i.v. infusion of human cell-free oxy-Hb and SNO-oxy-Hb (SNO-Hb)influenced muscle perfusion in the quadriceps of rats breathing room air(normoxia) was investigated. SNO-Hb decreased muscle perfusion more thanHb when compared to albumin (see FIG. 1A, p=0.055 and 0.3 vs. albumin,respectively; two-way ANOVA).

The pharmacological effects of these molecules in flank tumors ofnormoxic rats was then determined. In contrast to albumin, Hb induced arapid and sustained decrease in tumor blood flow (see FIG. 1B, p<0.01vs. albumin; two-way ANOVA). Unexpectedly, i.v. infusion of SNO-Hb alsoreduced perfusion (see FIG. 1C, p<0.05 vs. SNO-albumin; two-way ANOVA).Reductions in flow caused by oxy-Hb and SNO-Hb were not different(p>0.05; two-way ANOVA). However, the effects of the two Hbs were quitedifferent from the small increase in tumor perfusion that was observedfollowing albumin or SNO-albumin infusion i.v. (see FIGS. 1B and 1C).The effects of SNO-albumin and albumin were not significantly different(p>0.05; two-way ANOVA).

Example 2 Hyperoxia Prolongs the Half-life and Modulates the PressorActivity of Cell-Free SNO-Hb

It was reasoned that an increase in plasma oxygenation might stabilizeSNO-Hb, increase peripheral delivery, and thereby increase thebioactivity in tumors. Thus, whether the plasma half-life of SNO-Hb i.v.was prolonged in rats breathing 100% O₂ (hyperoxia) versus room air wasdetermined. Blood gas measurements showed that breathing 100% O₂ inducedsignificant increases in venous and arteriolar blood pO₂, pCO₂, andendogenous Hb oxygen saturation (HbO₂). Changes in Hb saturation wereidentical in femoral vein and tumor venules. Changes were lesspronounced in tumor-feeding arterioles versus the femoral artery, asexpected from blood deoxygenation along the arterial tree and at thetumor margin (see Table 1).

TABLE 1 Blood Gas and Hemoglobin Saturation Measurements. Room Air 100%O₂ Femoral artery: pO₂ (mm Hg) 82.4 ± 0.4 435.5 ± 34.7* pCO₂ (mm Hg)49.6 ± 1.3 56.9 ± 1.7* HbO₂ (%) 88.8 ± 0.4 98.2 ± 0.1* Femoral vein pO₂(mm Hg) 57.5 ± 1.2 69.8 ± 3.0* pCO₂ (mm Hg) 50.6 ± 1.7 72.0 ± 2.1* HbO₂(%) 67.7 ± 2.4 75.8 ± 3.4* Tumor artery HbO₂ (%) 76 ± 2 89 ± 2* Tumorvein HbO₂ (%) 69 ± 2 78 ± 3*

Due to low concentration (about 6.5 μmol/L) and alteration in thetetramer/dimer ratio as a function of concentration and O₂ saturation(dimer/tetramer equilibrium is 1000-fold lower in deoxy-Hb), it was notpossible to directly measure the oxygen saturation of exogenous Hbs.Instead, plasma SNO-Hb concentration versus time following infusionusing a DAF-2 assay was measured. The fitting of individual experimentaldata with exponential decay curves (R²=0.87±0.05 and 0.90±0.03 fornormoxic and hyperoxic conditions, respectively) revealed a 2-foldincrease in the half-life of the S-nitrosyl group in SNO-Hb in theplasma of hyperoxic versus normoxic rats (see FIG. 2, p<0.01; Student'st test). No influence of O₂ on the rate of SNO-Hb protein clearance wasobserved. Spectrophotometric measurements revealed that hyperoxia didnot affect the rate of protein clearance during the period of theexperiments (half-life about 30 minutes, independent of O₂ supply).Thus, these data suggest that the effects of O₂ are mediated by anallosteric mechanism that promotes the R structure.

The effects of hyperoxia alone on tumor and muscle perfusion ranged froma transient decrease that resumed within 5 minutes (i.e., before druginfusion) to no change. As expected from these observations, hyperoxiaabolished the reduction in tumor perfusion that was observed after i.v.infusion of SNO-Hb in normoxic rats (see FIG. 3A, p<0.005; two-wayANOVA). In contrast to SNO-Hb, native Hb reduced tumor perfusionindependently of O₂ supply (see FIG. 3B, p>0.05; two-way ANOVA).

Example 3 Hyperoxia Unmasks the Systemic Pressor Activity of Cell-FreeSNO-Hb

In order to identify the relative contribution of changes in vascularactivity versus systemic hemodynamics in the tumor perfusion response toSNO-Hb, i.v. infusion of SNO-Hb induced no significant change in thediameter of feeding arterioles in normoxic or hyperoxic rats asdetermined by window chamber experiments (see FIG. 4A, p>0.05; two-wayANOVA). Hyperoxia alone had no vascular effect (compare t=−5 to t=0 inFIG. 4A, white boxes). Interestingly, in the same rats, it wassimultaneously documented that SNO-Hb induced a potent decrease in tumorperfusion in normoxic animals, and that this decrease was prevented inhyperoxic rats. Thus, these results were identical to what was observedin flank tumors (see FIG. 3A).

Changes in tumor blood flow paralleled changes in the perfusion of theleg quadriceps muscle: the decrease in muscle blood flow after deliveryof SNO-Hb under normoxia was prevented under hyperoxia (see FIG. 4B,p<0.05; two-way ANOVA). In contrast, hyperoxia did not modify the effectof oxy-Hb in muscles.

Together, these observations suggested that SNO-Hb might indirectlymodulate tumor perfusion in an O₂-dependent manner as a consequence ofchanges in mean arterial pressure (MAP) and/or the heart rate (HR). Nochange in MAP (FIG. 4C) or HR (FIG. 4D) was observed when SNO-Hb wasinfused i.v. in normoxic animals (Student's t test versus values att=0). However, under hyperoxia, a significant increase in the MAP wasmeasured as soon as 10 minutes after the infusion of SNO-Hb i.v. (seeFIG. 4C, p<0.05 from t=+10 to t=+20 versus value at t=0; Student's ttest). There was no significant effect on HR (see FIG. 4D, p>0.05 forall values versus value at t=0; Student's t test). Interestingly, underthe same set of conditions, oxy-Hb had no effect on MAP during normoxiaor hyperoxia (see FIG. 4E, p>0.05 for all values versus value at t=0;Student's t test). Also, in contrast to SNO-Hb, oxy-Hb inducedbradycardia as soon as 5 minutes after infusion (see FIG. 4D, p<0.05from t=+5 to t=+20 versus value at t=0; Student's t test), which wasmore severe in hyperoxic than in normoxic rats. Hyperoxia alone inducedno significant changes in MAP (+1%) or HR (−3%; p>0.05, N=19; Student'st test).

Example 4 The Route of Administration Impacts the Pressor Activity ofCell-free SNO-Hb

Whether the physiological difference of blood oxygenation between veins(femoral vein infusion) and arteries (left carotid artery infusion) wassufficient to modulate the pressor activity of SNO-Hb was investigated.In normoxic animals, despite a trend towards preservation of the flow,the decrease in tumor perfusion after i.a. infusion of SNO-Hb was notsignificantly different compared to i.v. infusion (see FIG. 5A, p=0.09;two-way ANOVA). However, in contrast to i.v. SNO-Hb, the pressoractivity of i.a. SNO-Hb was not altered when this experiment wasrepeated in hyperoxic rats (p>0.05 versus SNO-Hb i.a. room air; two-wayANOVA). The lack of O₂ dependence contrasted with the strongO₂-dependent changes in tumor perfusion for i.v. infusions (these curvesare provided in FIG. 5A for comparison).

Furthermore, O₂ concentration in the breathing gas did not modify theperfusion of the quadriceps muscle when SNO-Hb was delivered i.a. (seeFIG. 5B, p>0.05; two-way ANOVA). As with the tumor results, the effectscontrasted with the changes observed following i.v. injection (see FIG.4B). Muscle blood flow remained unaltered after i.a. infusion of SNO-Hb,independently of O₂ delivery (see FIG. 5B).

MAP was highly sensitive to SNO-Hb provided i.a.; a significant increasein MAP as soon as 5 minutes and 10 minutes after drug delivery tonormoxic and hyperoxic rats, respectively was observed (see FIG. 5C,p<0.05 versus value at t=0; Student's t test). The degree ofhypertension, however, was unaffected by blood pO₂. In the same set ofexperiments, HR remained unchanged (see FIG. 5D). In controlexperiments, i.a. and i.v. infusion of albumin (used to monitor volumeeffects on the baroreceptor) did not induce any significant ordifferential change in tumor perfusion, MAP, or HR of tumor-bearing rats(all p>0.05 for albumin i.a. versus albumin i.v.; two-way ANOVA).

Example 5 Breathing ENO in Pure Oxygen Increases Tumor Perfusion

Tumors were implanted in rats as described hereinabove, and the ratswere set up to breathe either 100% O₂, 100% O₂ plus 100 parts permillion ethyl nitrite (ENO), normal room air, or normal room air plus100 ppm ENO. pO₂ was monitored in the tumor or in quadriceps muscle from30 minutes before the administration of the 100% O₂/100% O₂ plus 100 ppmENO/room air/room air plus 100 ppm ENO, during 30 minutes of breathingof the gases, and for 30 minutes after the administered gas supply wasremoved. The results of these experiments are presented in FIGS. 6Athrough 6D.

As shown in FIG. 6A, pO₂ in quadriceps muscle increased rapidly when the100% O₂ or 100% O₂ plus 100 ppm ENO was administered, and quicklyreturned to baseline when the breathing gas was turned off. As shown inFIG. 6B, this effect was not seen in rats breathing room air plus ENO.Measurements of pO₂ in tumors before, during, and after breathing 100%O₂ or 100% O₂ plus 100 parts per million ENO also showed an increase inpO₂ in tumors upon administration of the hyperoxic gas plus ENO,although the increase in pO₂ in rats breathing 100% O₂ alone wasinsufficient to raise the pO₂ to above 10 mm Hg (see FIG. 6C). FIG. 6Dshows that pO₂ remained largely unchanged in tumors in rats breathingroom air plus 100 ppm ENO changes in pO₂. It is interesting to note thatin rats breathing 100 ppm ENO plus 100% O₂, the pO₂ increased from lessthan 10 mm Hg to greater than 10 mm Hg, but the same increase was notseen in tumors of rats breathing ENO under normoxic conditions.

Example 6 Tumor Growth Delay after 100% Oxygen Plus ENO in Conjunctionwith Radiation

Tumors were implanted in rats as described hereinabove, and the tumorswere irradiated daily with 2 Gy of irradiation on days 0-4. Relativetumor volumes were determined daily from day 0 to day 18 in rats thatbreathed room air (N=4), 100% O₂ (N=4), or 100 ppm ENO plus 100% O₂. Asa control, relative tumor volumes for rats that breathed room air (N=3)and were not treated with radiation. were also determined.

The results are presented in FIG. 7. As can be seen, irradiated tumorsgrew more slowly in rats that breathed 100 ppm ENO plus 100% O₂ (▪) thanthey did in rats that breathed 100% O₂ (▴), rats that breathed room airalone (Δ), or non-irradiated tumors in rats that breathed room air (◯).

Discussion of Examples 1-6

S-nitrosylated Hb, when administered i.v. concomitantly with oxygenbreathing, is capable of maintaining tumor perfusion (i.e., itcounteracts the vasoconstrictive effects of both stroma-free Hb itselfand oxygen).

The presently disclosed subject matter thus relates in some embodimentsto creating S-nitrosylated hemoglobin in situ in the red blood cell,which yields the same effect, but without the concomitant difficultiesof dealing with a stroma-free Hb. To examine this concept, stroma-freehemoglobins, in the form of oxy-Hb and SNO-Hb were administered to tumorbearing rats during air and oxygen breathing, while tumor blood flow wassimultaneously monitored.

It was observed that i.v. infusion of human cell-free oxy-Hb and SNO-Hbinfluenced muscle perfusion in the quadriceps of rats breathing room air(normoxia). SNO-Hb decreased muscle perfusion more than oxy-Hb whencompared to albumin (see FIG. 1A). Studies were also performed intumor-bearing rats, where the tumors were transplanted to the flanks.The pharmacological effects of these molecules in tumors grown in theflank of normoxic rats were determined. In contrast to albumin, oxy-Hbinduced a rapid and sustained decrease in tumor blood flow (see FIG.1B). Unexpectedly, i.v. infusion of SNO-Hb also produced such a decrease(see FIG. 1C). Thus, the reductions in flow caused by oxy-Hb and SNO-Hbwere not different. However, the effects of the two Hb species werequite different from the small increase in tumor perfusion that wasobserved following albumin or SNO-albumin infusion i.v. (see FIGS. 1Band 1C).

The effects of the two hemoglobin species were quite different whenanimals were breathing oxygen during and following the infusion of thehemoglobin solutions. Hyperoxia prolonged the half-life and modulatedthe vasoactivity of cell-free SNO-Hb. Blood gas measurements showed that100% O₂ breathing induces significant increases in venous and arteriolarblood pO₂, pCO₂, and endogenous Hb oxygen saturation. Changes in Hbsaturation were found to be identical in femoral vein and tumor venules;it is less pronounced in tumor-feeding arterioles versus femoral artery,as expected from blood deoxygenation along the arterial tree and at thetumor margin (see Table 1).

The half life of the S-nitrosyl group of SNO-Hb was determined using theDAF-2 assay. Fitting of individual experimental curves with exponentialdecay curves (R²=0.87±0.05 and 0.90±0.03 for normoxic and hyperoxicconditions, respectively) revealed a 2-fold increase in the half-life ofthe S-nitrosyl group on SNO-Hb in the plasma of hyperoxic versusnormoxic rats (see FIG. 2). Of note, O₂ did not influence on the rate ofSNO-Hb protein clearance. The effects of hyperoxia alone on tumor andmuscle perfusion ranged from a transient decrease that resumed within 5minutes (i.e., before drug infusion) to no change. As expected fromthese observations hyperoxia abolished the reduction in tumor perfusionafter i.v. infusion of SNO-Hb in normoxic rats (see FIG. 3A). Incontrast to SNO-Hb, oxy-Hb reduced tumor perfusion independently of O₂supply (see FIG. 3B).

A further discovery showed that hyperoxic gas breathing unmasked asystemic pressor activity of cell-free SNO-Hb. In window chambers, i.v.infusion of SNO-Hb induced no change in diameter of tumor-feedingarterioles in normoxic or hyperoxic rats (see FIG. 4A). Additionally,hyperoxia alone had no discernable effect on arteriolar diameter. Inspite of the lack of tumor arteriolar effects, SNO-Hb potently induced adecrease in tumor perfusion in normoxic animals, and this decrease wasprevented in hyperoxic ones. These results were identical to what wasobserved in flank tumors (see FIG. 3A). The lack of change in arteriolardiameter in tumor arterioles suggested that the vasoactive properties ofSNO-Hb were either occurring upstream or that systemic effects dominatedthe change in flow within the tumors. Results in muscle helped todiscern the underlying mechanism.

Changes in the tumor blood flow were paralleled by changes in theperfusion of the leg quadriceps muscle: a decrease in muscle blood flowafter delivery of SNO-Hb under normoxia was prevented under hyperoxia(see FIG. 4B). In contrast, hyperoxia did not modify the effect ofoxy-Hb in muscle perfusion. Altogether, these observations led to theconclusion that SNO-Hb indirectly modulated tumor perfusion as aconsequence of changes in the mean arterial pressure (MAP) and/or theheart rate (HR), in an O₂-dependent way. No changes in MAP (see FIG. 4C)or HR (see FIG. 4D) were observed when SNO-Hb was infused i.v. innormoxic animals. However, under hyperoxia, a significant increase inthe MAP as soon as 10 minutes after the infusion of SNO-Hb i.v. wasobserved (see FIG. 4C). There was no significant effect on HR (FIG. 4D).Interestingly, under the same conditions, oxy-Hb had no effect on MAPduring normoxia or hyperoxia (see FIG. 4E). Moreover, in contrast toSNO-Hb, oxy-Hb induced bradycardia as soon as 5 minutes after infusion(see FIG. 4D), which was more severe in hyperoxic than in normoxic rats.Hyperoxia alone induced no significant changes in MAP (+1%) or HR (−3%).

The O₂ dependency of the bioactivity of SNO-Hb prompted determination ofwhether the physiological difference of blood oxygenation between veins(femoral vein infusion) and arteries (left carotid artery infusion) wassufficient to modulate the vasoactive properties of SNO-Hb. In normoxicanimals, i.a. infusion of SNO-Hb induced a less dramatic decrease intumor perfusion than when the drug was delivered i.v. (see FIG. 5A). Incontrast to the i.v. case, the vasoactive effects of i.a. SNO-Hb werenot modified when this experiment is repeated in hyperoxic rats. Thelack of O₂ dependency contrasted with the strong O₂-dependent changes intumor perfusion for i.v. infusions (these curves are provided in FIG. 5Afor comparison). Moreover, O₂ supply did not modify the perfusion of thequadriceps muscle when SNO-Hb is delivered i.a. (see FIG. 5B). As withthe tumor results, the effects contrasted with the changes that wereobserved following i.v. injection (see FIG. 4B). Muscle blood flowremained unaltered after i.a. infusion of SNO-Hb, independently of O₂delivery (see FIG. 5B). However, MAP was highly sensitive to SNO-Hbi.a.: a significant increase in MAP was observed 5-10 minutes afterSNO-Hb delivery to normoxic and hyperoxic rats, respectively (see FIG.5C). The degree of hypertension, however, was unaffected by blood pO₂.In the same set of experiments, HR remained unchanged (see FIG. 5D).

These data revealed interesting biological properties of Hb in theplasma. First, the effects of SNO-Hb were dependent on blood oxygenation(biologically relevant range) while the effects of oxy-Hb were not.Second, when delivered in oxygenated blood, SNO-Hb affected the centralcontrol of hemodynamics.

During hyperoxia, SNO-Hb stabilization occurred in the venouscirculation. Evidence for this came from the fact that that SNO-Hbbioactivity was identical following hyperoxic i.v. and normoxicintraarterial (i.a). infusions. Moreover, hyperoxia combined with i.a.infusion did not further modulate the effects of SNO-Hb. Changescorrelated with increased blood pO₂ but not pCO₂. Hence, physiologicallyoxygenated blood at the site of delivery was sufficient to prolongSNO-Hb bioactivity with maximal efficiency.

SNO release in oxygenated plasma cannot be completely prevented,however. Indeed, i.a. infusion during normoxia and i.v. and i.a.infusions during hyperoxia demonstrated a central pressor activity ofSNO-Hb whereas oxy-Hb (hyperoxia, i.v.) did not show this effect.Baroreceptor activity opposes increases in MAP and resistance to flow byinhibiting the sympathetic nerve activity. At physiologicalconcentrations, hemodynamic responses to oxy-Hb apparently activated thebaroreceptor that buffers changes in MAP by decreasing HR. Theobservation that SNO-Hb in oxygenated blood raised the MAP withoutaltering perfusion or HR was therefore unexpected. Comparison indicatedthat the systemic pressor activity of SNO-Hb resulted from a directinhibition of the baroreceptor reflex.

NO is well known, as a neurotransmitter, to regulate baroreceptoractivity through sympathetic afferent nerve fibers (Paton et al., 2001).In contrast, circulating NO donors have no direct inhibitory action onthe baroreceptor unless delivered at high concentration (≧100 μmol/L)directly into the carotid sinus, and the baroreceptor activity recoversspontaneously within seconds. This contrasts with the prolongedinhibition of the baroreceptor disclosed herein in the presence ofSNO-Hb. Unlike classical NO activity, SNO-Hb bioactivity was mediated byNO-related species distinct from NO itself (e.g., small molecular weightSNO), and these species would then have different effects than NO gas.

SNO conjugation successfully overcame the pressor activity of micromolarconcentrations of Hb in hypoxic (tumors) and normoxic tissues. Thisactivity required injection in hyperoxygenated venous blood or normoxicarterial blood. Under such circumstances SNO-Hb was allostericallystabilized. The peripheral effects of SNO-Hb were a composite betweencentral activity (baroreceptor inhibition) and allostery-facilitatedactivity in the periphery. In contrast, native Hb at concentrationscharacteristic of hemolytic states had no central pressor effect.

Thus, the presently disclosed subject matter provides improved deliveryof oxygen to tumors while preserving perfusion. The combination ofimproved oxygenation and perfusion maintenance can translate intoincreased tumor radioresponsiveness and chemoresponsiveness. Increasedlevels of SNO-Hb in the peripheral circulation can prevent thevasoactive effects of hyperoxic gases, thereby maintaining and evenimproving perfusion and oxygen delivery to hypoxic tumor regions.Unloading of SNO from Hb in the more hypoxic regions of a tumor can alsoserve to radiosensitize and/or chemosensitize such regions. In someembodiments, the presently disclosed subject matter is based at least inpart on modifying endogenous stores of SNO-Hb. In some embodiments, thepresently disclosed subject matter provides S-nitrosylated-Hb directlyto the subject.

Vasoconstriction remains a major limitation in the clinical use ofHb-based blood substitutes. Cell-free Hb preparations are devoid of theSNO that normally serves to counter their vasoconstrictive effects invivo. Disclosed herein is the discovery that SNO reconstitution of Hbcan reverse the vasoconstrictor activity of oxy-Hb, and further thatSNO-Hb can be manipulated allosterically to maximize O₂ delivery. Inrats breathing room air (normoxia), SNO-Hb induced a greater decrease intissue perfusion than native Hb (see FIG. 1A and Table 2). These resultssuggested that oxy-Hb and SNO-Hb operated by different mechanisms. Whilethe effects of oxy-Hb could be readily explained by NO scavenging (the“oxyhemoglobin,” or met-Hb-forming reaction), those of SNO-Hb wereconsistent with other NO donors, which paradoxically also decreasetissue perfusion. NO-mediated dilation of healthy blood vessels upstreamof tumors creates shunts that divert blood away from the tumors(vascular steal). By raising the concentration of the inhaled O₂, SNO-Hbbioactivity can be targeted to more distally blood vessels, and elicitsimprovements in tumor blood flow.

TABLE 2 Hemodynamic Properties of Cell-free Human SNO-Hb and Oxy-HbTUMOR vascular SYSTEMIC perfusion diameter perfusion MAP HR SNO- IV Roomair  ↓ * 0 ↓ 0 0 Hb 100% O₂ 0 0 0 ↑ 0 IA Room air 0 nd 0 ↑ 0 100% O₂ 0nd 0 ↑ 0 Oxy- IV Room air ↓ nd ↓ 0 0/↓ HB 100% O₂ ↓ nd ↓ 0 0/↓ * refersto changes versus values before treatment: 0, unchanged; ↓, decreased;↑, increased; nd, not determined. SNO-Hb, human S-nitrosohemoglobin;oxy-Hb, human oxyhemoglobin; IV, intravenous infusion; IA,intra-arterial infusion; MAP, mean arterial pressure; HR, heart rate.

To further test oxygen-dependent regulation of NO release, the activityof cell-free SNO-Hb versus native Hb in tumors was investigated. Thewell-characterized R3230Ac rat mammary tumor model was employed toobserve low but biologically significant pO₂ conditions. In normoxicrats, SNO-Hb i.v. lowered perfusion in tumors to the same extent as inmuscles (see Table 2). In tumors, oxy Hb exhibited a similar decrease inperfusion, while albumin (and to a similar degree SNO-albumin) tended toincrease perfusion slightly (see FIGS. 1B and 1C).

Interestingly, the SNO-Hb-induced decrease in tumor perfusion was notassociated with tumor-feeding vessel constriction or changes in MAP orHR (see Table 2). While it is not desired to be bound by any particulartheory of operation, it appears that the observations can be attributedto: (1) vasoconstriction (NO scavenging) of microvessels within thetumor; and/or (2) vasodilation (SNO release) of vessels in paralleltissues that creates a vascular steal. Because of ongoing angiogenesis,most vessels downstream of feeding arterioles in fast-growing rodenttumors lack structural elements for vasoactivity, and they lackfunctional endothelial NO synthase. Hence, during normoxia, SNO-Hb i.v.must decrease tumor perfusion through vascular steal. This is consistentwith the previous observation that, although oxy-Hb provoked a decreasein healthy muscle perfusion (as expected from a NO scavenger), itincreased perfusion in muscle surrounding the R3230AC tumor (a NOdonor-like response) consistent with a steal effect.

To gain further mechanistic insight, whether manipulation of bloodoxygenation would impact SNO-Hb bioactivity was tested. Normobaric 100%oxygen breathing (hyperoxia) induced a significant increase in venousand arteriolar pO₂ (see Table 1). Although the delivery of pure oxygensometimes reduced tumor and muscle perfusion (see FIGS. 3A, 3B, 5A, and5B between t=−5 and t=0), this effect was transient and returned tobaseline within less than 5 minutes. Thus, the vasoactive properties ofoxygen per se did not affect the response to SNO-Hb or oxy-Hb. Upon i.v.infusion in hyperoxic animals, SNO modified Hb successfully opposed thereduction in perfusion created by Hb in tumors (compare FIGS. 3A and3B). Increases in MAP were seen in response to SNO-Hb during 100% O₂breathing, but there was no change in tumor/muscle perfusion or HR (seeTable 2). By comparison, the reduction in tumor perfusion by native Hbremained unaffected by hyperoxia (see FIG. 3B). This is likely accountedfor by lowered systemic perfusion and/or bradycardia, not by changes inMAP (see Table 2).

Hb dissociation from tetramers to dimers depends on Hb concentration andoxygen tension, and NO release from dimers is unresponsive to allostericeffectors (e.g., O₂). Assuming a plasma concentration of about 6.5μmol/L, about 35% of SNO-Hb would be tetramers (K_(D) for R and Tconformers are 3 μmol/L and 3 nmol/L, respectively) at room air and theamount would increase precipitously as O₂ tension declines—the majoritywould be tetramers at tissue pO₂. Is there enough tetrameric SNO-Hb tosense oxygen and regulate NO delivery? Evidently yes, as a finely tunedregulation of SNO-Hb bioactivity by oxygen tension (NO release dependson local pO₂) that contrasted with the O₂-independent behavior of oxy-Hbwas observed. That the plasma half-life of SNO on Hb was more thandoubled during hyperoxia while protein clearance remained unaffected wasalso observed. Thus, the prolonged half-life of SNO bound to equallystable Hb proteins supported the assertion that SNO release by SNO-Hb isdisfavored under hyperoxia. Collectively, these data provided adefinitive demonstration of allosteric regulation by O₂ of NO deliveryfrom SNO-Hb

During hyperoxia, SNO-Hb is greatly stabilized, surviving arteriovenoustransit. SNO-Hb bioactivity was thus indistinguishable followinghyperoxic i.v. and normoxic i.a. infusions (see Table 2). To address apoint of confusion, it is noted that SNO-Hb will exert activity at bothhigh and low pO₂ but that this activity will be potentiated at low pO₂.Effects will therefore manifest locally. Consistent with thisinterpretation, hyperoxia had no effect on i.a. infusions of SNO-Hb butmarkedly altered i.v. responses (see Table 1).

On close inspection, the data disclosed herein unravel a central effectof SNO-Hb on control of hemodynamics. An increase in MAP was observedfollowing i.a. infusion during normoxia, and both i.v. and i.a.infusions during hyperoxia. Native Hb (hyperoxia, i.v.) did not produceincreases in MAP (see Table 2) under any condition. Baroreceptoractivity opposes increases in MAP and resistance to flow. Native Hbactivated the baroreceptor, which opposed changes in MAP by decreasingthe HR. In contrast, strikingly, SNO-Hb raised the MAP without alteringperfusion (in the organs that were monitored) or HR. The systemicpressor activity of SNO-Hb resulted from a direct inhibition of thebaroreceptor reflex.

Thus, SNO-Hb activity in hyperbaric hyperoxia is sufficient to inhibitthe baroreceptor. As a neurotransmitter, NO is known to regulatebaroreceptor activity through sympathetic afferent nerve fibers.Exogenous NO has an inhibitory action on the baroreceptor, albeit onlyat high concentrations, and under such circumstances that thebaroreceptor activity recovers within seconds. Unlike classical NOactivity, SNO-Hb bioactivity is mediated by species distinct from NO⁻itself (e.g., low-mass SNOs). It is noteworthy that other endogenousS-nitrosothiols such as SNO-cysteine can suppress baroreceptor activityindependently of cGMP generation (the mediator of classicalnitrovasodilator activity). Stereoselective recognition sites in thebaroreceptor vasculature could mediate baroreceptor inhibition byS-nitrosylated species such as SNO-cysteine and SNO-Hb.

In conclusion, disclosed herein is the observation that SNOreconstitution of Hb successfully overcame the reduction in tumorperfusion created by Hb itself. This activity involved the allostericcontrol of NO release by O₂. The hemodynamic effects of SNO-Hb were acomposite of central activity (baroreceptor inhibition) andallostery-facilitated NO release in the peripheral circulation. Incontrast, native Hb at concentrations characteristic of hemolytic stateshad no central pressor effect. These results are summarized in Table 3.

TABLE 3 Bioactivity of Cell-free SNO-Hb and Oxy-Hb. Vascular pO₂Peripheral Baroreceptor at injection site NO-related chemical fateeffect effect

low high Met-Hb + iron-nitrosyl Hb SNO-Hb vasoconstrictionvasodilation^(o) none inhibition

any Met-Hb + iron-nitrosyl Hb vasoconstriction none SNO-Hb,S-nitrosohemoglobin; oxy-Hb, oxyhemoglobin; NO, nitric oxide; Met-Hb,methemoglobin. ^(o)Dose-dependent effect.

While the concentration of Hb used was low from the O₂ deliverystandpoint, it greatly exceeded the concentration of any endogenous NO.In light of the results presented herein, development of safe Hb-basedblood substitutes might include approaches that not only preserve Hballostery (e.g., crosslinking to prevent dissociation into dimers) butalso reconstitute SNO content. Reoxygenation of hypoxic tissues mightbenefit from both the central pressor and blood flow increasing effectselicited by SNO-Hb. Further, the treatment of trauma or septic patientsmight involve manipulation of SNO-Hb allostery to limit excessive NOrelease.

Materials and Methods for Examples 7-10

Tumor implantation, growth, and definition of treatment size. Cells aregrown to 80% confluency in culture dishes and implanted into the flanksof athymic nude mice at a concentration of 1 million cells per animal.The tumor volume is measured starting from the day of palpableappearance. Tumor volume is calculated as volume (mm³) equals (p/6)xy²,with x=longer and y=shorter axis of the tumor (see Baumann et al.,1990). Animals with tumors of a size of 500 mm³, or an average diameterof 10±1 mm, are considered eligible for treatment.

Administration of gas to mice. Gas mixtures are administered overcustomized face masks using mixture-specific mass flow controllers(provided by NITROX LLC, Durham, N.C., United States of America). Fiveanimals are ventilated at a time using a one-in-five distributor. Duringall measurements equal body temperature is maintained by placing theanimals on metal plates that sit on water-circulated heating pads.

Radiation treatment of tumors. Tumor irradiation is performed in using aclinical linear accelerator. Animals are anesthetized at the site ofirradiation using pentobarbital. Flank tumors are exposed usingcustomized animal restrainers. The animal body is shielded using leadblocks.

Measurement of tumor oxygenation using fiber-optic sensors. Tumoroxygenation is measured using the Oxylite system (Oxford Optronix,Oxford, United Kingdom), which is based on probe-based measurements ofoxygen-dependent fluorescence quenching and signal detection overfiber-optic cables. The system employs concurrent measurements of tissuetemperature using thermocouples.

Example 7 Optimization of ENO Dose

The lowest effective dose (LED) of ENO is determined on FaDu (humanhypopharyngeal carcinoma cells, available from the American Type CultureCollection, Manassas, Va., United States of America) tumor xenographswith tumor oxygenation as an endpoint. Separate cohorts of animals areexposed to room air, O₂ alone, or O₂ supplemented with differentconcentrations of ENO (e.g., 100 ppm, 75 ppm, 50 ppm, 25 ppm, 10 ppm).Each group includes 15 animals. Oxygenation is measured using theOxylite system and by histology comparing pimonidazole hypoxic fractionwith total vital tissue. The endpoint is the identification of thelowest ENO dose showing improvement in tumor oxygenation equivalent orbetter than that achieved with a 30 minute continuous exposure of 100ppm ENO+O₂, which as disclosed herein increased tumor pO₂ above 10 mmHg.Nitrite/nitrate levels (a follow up product of NO) in the blood ofanimals treated with the LED are analyzed to assess systemic effects ofthis dose of ENO. As a surrogate marker of ENO exposure and to validatethe mechanistic action of ENO, cGMP levels are also assessed in thetumor.

More particularly, FaDu cells are implanted into athymic nude mice andgrown to treatment size. Animals are anesthetized with nembutal,catheterized for re-dosing of anesthesia, and placed on a heated animalrestrainer. In order to simulate irradiation conditions, the animals areplaced on a clinical irradiator for the duration of the experiment. Twooxygen probes are inserted into different parts of the tumor: one closeto the surface, the other at the center of the tumor. A third probe isinserted into the muscle of the opposite hind leg, as a control.Thermocouples are inserted into the tumor and the muscle. Oxygen andtemperature values are monitored constantly throughout the experiment. Alaser Doppler probe (OxyFlo, Oxford Optronix, Oxford, United Kingdom) isinserted into the tumor to measure changes in tumor blood flow duringtreatment.

Room air is administered to the animal at a rate of 5 L/minutes via aface mask. After 30 minutes, room air is replaced with an ENO/oxygen gasmixture, administered at the same rate. At the same time, pimonidazoleis administered i.p. at a concentration of 20 mg/kg. The animal isallowed to breathe the gas mixture for 60 minutes. Five minutes beforesacrificing the animal, Hoechst 33342 (Bisbenzimide, Sigma, St. Louis,Mo., United States of America) is injected i.v.

The tumor is excised and snap frozen in liquid nitrogen. Samples aresectioned and analyzed for pimonidazole hypoxic fraction as outlinedhereinbelow. The first ENO dose tested is 25 ppm. Depending on whetherthis dose is already sufficient not to increase the tumor oxygenation toat least 10 mmHg, continued testing with the next lower (10 ppm) or thenext higher (50 ppm) dose is performed. The endpoint of this study isthe identification of the lowest effective dose (LED), which is thelowest dose of ENO showing improvement of tumor oxygenation beyond 10 mmHg.

Five animals out of each group are subjected to treatment as outlined,with the exception that after removal of the tumor, a blood sample isobtained by cardiac puncture from the anesthetized animal. Each bloodsample (approx. 500 μl) is mixed 1:5 with a nitrite/nitrate preservationsolution (see Dejam et al., 2005). Analysis of nitrite/nitrate isperformed. The endpoint tests whether the nitrite/nitrate levels foundin the blood plasma of the animals correlated with (and thus aredetermined by) the administered ENO concentration.

All tumor samples are also analyzed for cGMP content, an intracellulardownstream effector of NO. Slices from tumor samples (consecutive tothose analyzed for pimonidazole) are pooled into lysis buffer, subjectedto extraction, and analyzed for cGMP using an antibody-based commercialmethod (cGMP BIOTRAK™ enzymeimmunoassay system, Amersham Pharmacia,Piscataway, N.J., United States of America). This allows for directcomparison of cGMP values (indicating the presence of NO) and the extentof pimonidazole hypoxic fraction in the tumors.

Example 8 Determination of Single Dose Radiation Dose Modifying Factor(DMF) for ENO Plus Oxygen

The relative value of a radiosensitizer to improve radiation response isquantified by determining the Dose Modifying Factor (DMF). This isdetermined by establishing the ratio of radiation doses required to cure50% of tumors for radiation alone divided by the dose required forradiation plus radiosensitizer (see Baumann et al., 1990). Tumor-bearinganimals with tumors grown to a specified size range are randomized toreceive pre-defined single doses of radiation (that bracket the dosesexpected to yield between 10 and 90% local tumor control) ±ENO+O₂breathing or O₂ breathing alone. The ENO dose optimized as in EXAMPLE 7is used. The radiation dose that leads to a lack of regrowth in 50% ofthe tumors within the observation period (TCD₅₀) for each treatment armis determined using logistic regression methods. It is expected that theDMF will be greater than one and will be largest for ENO+O₂ comparedwith O₂ alone.

More particularly, FaDu tumors are grown in nude mice. Starting from theday of palpable tumor appearance, tumor growth is monitored biweeklyusing caliper measurements by an investigator who is blinded to thegrouping of the animals. When tumors reach treatment size, radiation isapplied as a single dose in the range between 15 to 45 Gy, with 5 Gyincrements and eight animals per group. Treatment arms include airbreathing+irradiation, pure oxygen breathing plus irradiation, andENO/O₂ optimized dose (as determined in EXAMPLE 7) plus irradiation. Gasflow rates are equivalent to those specified in EXAMPLE 7. Muscle pO₂ ismonitored during radiation treatment using Oxylite probes placed in themuscle of the tumor-free leg to ensure consistency in ENO exposure.After treatment, animals are monitored for tumor regression andregrowth. Animals that show regression are followed for 120 days. Thosethat do not show regrowth in that time interval are considered to haveachieved local tumor control. The different treatment modalities arecompared with each other by calculating the TCD₅₀. The Dose-ModifyingFactor (DMF) is the ratio of the TCD₅₀ values of the treatment arms thatare compared (see Yaromina et al., 2005). The DMF of ENO/O₂ and roomair, and ENO/O₂ and pure oxygen breathing is determined.

Example 9 Pathological Consequences of Irradiation Administered with andwithout ENO+Oxygen Breathing

To explore the pathologic consequences of ENO/O2 breathing duringirradiation (using a dose near the TCD₅₀ as determined in EXAMPLE 8),the effect of this treatment on hypoxic fraction, as assessed by hypoxiamarker uptake, proliferation and apoptosis rates, percentage of necroticarea, and vascular density is assessed. These parameters are measured,using quantitative immunohistochemistry, at defined time points afterradiation treatment.

More particularly, to explore the pathologic consequences ofco-treatment of ENO/O₂ breathing plus irradiation, the effect of thesetreatments on the following parameters is determined:

(1) hypoxic fraction, as assessed by hypoxia marker uptake

(2) proliferation and apoptosis rates

(3) percentage of necrotic area

(4) vascular density

For these experiments, separate cohorts of animals are studied. Tumorsare grown to treatment size and then receive a single radiation dosethat is below that required to cure tumors in any of the treatmentgroups, but that is sufficient to cause growth delay by radiation alone.Out of each treatment group (room air, pure oxygen, ENO best dose,untreated) 12 animals are removed after treatment in a time dependentmanner: animals are removed in triplicate at 0, 24, 48, and 72 hoursafter treatment, respectively. All of these animals are injected withpimonidazole at 6 μl/g from a stock solution of 10 mg/mL 1.5 hoursbefore sacrifice. Hoechst 33342 is injected into each animal's tail vein5 minutes before tumor removal to serve as a perfusion marker.

Tumors are cryosectioned and stained for the degree of hypoxia(pimonidazole), proliferation activity (KI67), apoptosis (DNA strandbreakage, TUNEL), CD31 for microvessel density, perfused microvesseldensity (co-localization of Hoechst 33342 and CD31), and necrosis (basedon histologic assessment of H/E stained tissues) as follows:

Pimonidazole Hypoxic Fraction. Pimonidazole is a well-establishedimmunohistochemical marker of tumor hypoxia. Tumors are cryosectionedand sections fixed and stained with fluorescently-labeled antibodies.Slides are scanned using a fluorescence microscope equipped with acomputerized stage and shutter. After fluorescence imaging, the samesections are stained with hematoxylin/eosin to determine vital tumorareas. The endpoint of these measurements are the differences in thepimo-positive vs. overall vital tumor area between treatment groups 2and 1 as shown in Table 4.

TABLE 4 Treatment Groups and Dosing Schedule for EXAMPLE 8 Radiationdose Flow rate (L/minutes) # # Treatment group (Gy) ENO Oxygen Nitrogenmice 1 ENO (LED) 25 TBD TBD TBD 12 2 Pure oxygen 25 — 4.5 0.5 12 3 Roomair 25 — 1.05 3.95 12 4 No treatment — — — — 12

Percentages of vital tissue that are positively stained for pimonidazoleare compared between the treatment groups. Differences between groupsare tested for significance using standard ANOVA methods. Tumors arecryosliced and sections are fixed and stained with fluorescence-labeledantibodies. Slides are scanned using a fluorescence microscope withcomputerized stage and shutter, and, after imaging, stained and imagedfor hematoxylin/eosin to determine vital tumor areas.

The KI67 proliferation index. The nuclear antigen KI67 is detected usingestablished immunohistochemical protocols involving a polyclonal primaryand fluorescently-labeled secondary antibody and a Hoechst 33342counterstain to identify cell nuclei. The KI67 positive area isevaluated using a 16 bit microscope camera and grayscale analysis asdescribed in Moeller et al., 2005.

Apoptosis. Detection of the percentage of apoptotic cells by terminaltransferase dUTP nick end labeling (TUNEL) is performed usingestablished protocols (see e.g., Le et al., 2005). The signal isvisualized using fluorescence microscopy and evaluated as describedhereinabove.

Quantification of microvessel density. Tumor microvessels are detectedby CD31 staining as described in Peeters et al., 2004, followed byfluorescence-based detection of the primary antibody. The slides arescanned under a fluorescence microscope with a motorized travelingstage. Images are background-corrected and filtered and microvesseldensity is determined in selected areas using particle counts andhistogram analysis.

Perfused microvessel density. This is determined by examining thepercentage of CD31 positive vessels that exhibit perivascular Hoechst33342 staining.

Quantification of percent necrosis. This is determined using imageanalysis software. Total tumor area is identified and quantified interms of mm² surface area. The percentage area that is necrotic isdetermined by using planimetry to outline necrotic regions. Thepercentage of necrotic area is calculated as the ratio of the totaltumor area divided into the area of tissue that is necrotic. Areas oftissue that contain artifacts, such as tearing during sectioning, aresubtracted from the overall tumor area.

Example 10 Ability of Optimized ENO+Oxygen Dose to Improve TumorOxygenation and Reduce Hypoxia

Measurements of oxygen concentration and perfusion are made prior to andduring breathing of ENO+oxygen, oxygen alone, or air alone. Using dosefinding experiments, the degree of variability of oxygenation responseto ENO+oxygen breathing among tumors with different metaboliccapabilities is assessed.

In order to assess the heterogeneity of tumor response to ENO+oxygenbreathing, FaDu tumors are compared to SiHa (human cervical cancer) andWiDr (human colorectal cancer) xenografts. These tumor lines have beenchosen in part because that these tumors differ in their intrinsic ratesof oxygen consumption, and differences in oxygen consumption are likelyto have an impact on the efficiency of strategies to deliver oxygen tothe tumor. Measurements of tumor oxygenation are performed with theOxford Optronix fluorescence quenching probe paralleled by Laser Dopplermeasurements (OxyFlo, Oxford Optronix, Oxford, United Kingdom),providing information on whether changes in oxygenation are related totumor blood flow changes. Dose finding experiments are performed on SiHaand WiDr tumors as described in EXAMPLE 7. The ENO LED identified forFaDu tumors in EXAMPLE 7 is used as a starting point, and increases ordecreases from this dose in 25 ppm increments depending on whether ornot oxygenation response is lower or higher in FaDu tumors, are employed(see e.g., an exemplary dosing schedule presented in Table 5). Theoxygenation effect in FaDu tumors is also confirmed.

TABLE 5 Treatment Groups and Dosing Schedule ENO Flow rate (L/minutes) #mice # mice LED_(FaDu) +/− ENO Oxygen Nitrogen WiDr SiHa 100 ppm  TBD4.5 — 10 10 75 ppm TBD 4.5 TBD 10 10 50 ppm TBD 4.5 TBD 10 10 25 ppm TBD4.5 TBD 10 10  0 ppm TBD 4.5 TBD 10 10 Pure — 4.5 0.5 10 10 oxygen Roomair — 1.05 3.95 10 10

REFERENCES

The references listed below as well as all references cited in thespecification, including patents, patent applications, and journalarticles, are incorporated herein by reference to the extent that theysupplement, explain, provide a background for, or teach methodology,techniques, and/or compositions employed herein.

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

1. A method of enhancing delivery of a diagnostic, therapeutic, orprognostic agent to a hypoxic tumor in a subject, the method comprising:(a) administering to the subject a nitrosylation agent that inducesnitrosylation of hemoglobin in the subject, (b) administering to thesubject a hyperoxic gas, and (c) administering the diagnostic,therapeutic, or prognostic agent to the subject, wherein delivery of thediagnostic, therapeutic, or prognostic agent to the tumor in the subjectis enhanced.
 2. The method of claim 1, wherein the diagnostic,therapeutic, or prognostic agent comprises an imaging agent.
 3. Themethod of claim 1, wherein the hyperoxic gas is selected from the groupconsisting of 100% oxygen and carbogen.
 4. The method of claim 1,wherein the subject is a mammal.
 5. The method of claim 4, wherein themammal is a human.